A Pleiotropic Role for the Orphan Nuclear Receptor Small Heterodimer Partner in Lipid Homeostasis and Metabolic Pathways

Nuclear receptors (NRs) comprise one of the most abundant classes of transcriptional regulators of metabolic diseases and have emerged as promising pharmaceutical targets. Small heterodimer partner (SHP; NR0B2) is a unique orphan NR lacking a DNA-binding domain but contains a putative ligand-binding domain. SHP is a transcriptional regulator affecting multiple key biological functions and metabolic processes including cholesterol, bile acid, and fatty acid metabolism, as well as reproductive biology and glucose-energy homeostasis. About half of all mammalian NRs and several transcriptional coregulators can interact with SHP. The SHP-mediated repression of target transcription factors includes at least three mechanisms including direct interference with the C-terminal activation function 2 (AF2) coactivator domains of NRs, recruitment of corepressors, or direct interaction with the surface of NR/transcription factors. Future research must focus on synthetic ligands acting on SHP as a potential therapeutic target in a series of metabolic abnormalities. Current understanding about the pleiotropic role of SHP is examined in this paper, and principal metabolic aspects connected with SHP function will be also discussed.


Introduction
Nuclear receptors (NRs) constitute a unique family of ligandmodulated transcription factors. NRs mediate cellular response to small lipophilic endogenous and exogenous ligands [1,2] and are responsible for sensing a number of hormones, including steroid and thyroid hormones, and act as positive and negative regulators of the expression of specific genes [3][4][5]. Therefore, NRs play a central role in many aspects of mammalian development, as well as lipid homeostasis, physiology, and metabolism. NRs make up one of the most abundant classes of transcriptional regulators in the body and have emerged as promising pharmaceutical targets.
Classically, NRs consist of several functional domains, that is, a variable N-terminal ligand-independent transactivation domain (which often exhibits a constitutive transcription activation function (AF-1)), a highly conserved DNAbinding domain (DBD) that contains two zinc fingers, a hinge domain (a variable linker region), and a multifunctional C-terminal domain. Furthermore, the C-terminal domain includes the ligand binding (LBD), the dimerization interface, and the ligand-dependent transactivation domain AF-2 [1,6].
Small heterodimer partner (SHP; NR0B2 for nuclear receptor subfamily 0, group B, member 2; MIM number 604630, 601665) is a member of the mammalian NR superfamily, due to the presence of a putative ligand-binding domain (LBD) [7]. SHP functions as a corepressor through heterodimeric interaction with a wide array of nuclear receptors and repressing their transcriptional activity. SHP achieves its goal via several members of the NR superfamily that are able to regulate SHP expression. However, SHP is also a unique and atypical NR because it lacks the classical DNA-binding domain (DBD), generally present in other NRs [8]. The NR0B family of NRs consists of 2 orphan receptors: SHP and DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1). DAX1 is a gene whose mutation causes the X-linked adrenal hypoplasia congenita [9] and is the only family member that lacks a conventional DBD. DAX-1 (NR0B1) is therefore seen as the closest relative of SHP in the NR superfamily [10][11][12]. Both SHP and DAX-1 appear to be specific to vertebrates. In this respect, no homologous genes have been found in Drosophila melanogaster or Caenorhabditis elegans [12]. Whereas SHP is different from other conventional NRs both structurally and functionally, it acts as a ligand-regulated receptor in metabolic pathways [13]. SHP belongs to the orphan subfamily since there is no known ligand for this receptor, except for some retinoid-related molecules [14]. SHP inhibits transcriptional activation by working on several other nuclear receptors, that is, directly modulating the activities of conventional nuclear receptors by acting as an inducible and tissue-specific corepressor [12,15]. The discovery of SHP dates back to 1996 [10]; since then, this orphan NR has been identified as a key transcriptional regulator of signaling pathways [8,16] involving fundamental biological functions and metabolic processes. Such processes include cholesterol, bile acid and fatty acid metabolism, glucose and energy homeostasis, and reproductive biology [17]. Experiments performed by fluorescence in situ hybridization (FISH) analysis of the human metaphase chromosome have shown that SHP is found at a single locus on chromosome 1 at position 1p36.1 and consists of two exons and a single intron spanning approximately 1.8 kb with 257 amino acids in humans [18]. In mice and rats, SHP resides on chromosomes 4 and 5, respectively, both consisting of 260 amino acids. SHP expression is predominantly observed in the liver [10,18], but it is also detected at lower levels in other tissues, including the pancreas, spleen, small intestine, colon, gallbladder, kidney, adrenal gland, ovary, lung, brainstem, cerebellum, heart, and thymus (Table 1) [19][20][21].
Gene expression of SHP is regulated by several factors including NRs, transcription factors, and a number of additional conditions and substances, as extensively reported in Table 2. Also, the central role of SHP is clear since this NR is able to act as a coregulator for wide range of targets, namely, NRs/transcription factors/transcriptional coregulators and few different molecules, as depicted in Table 3. In general, SHP acts as a repressor of the transcriptional activity of the specific interacting partner (via LBD of the partner and NR boxes of SHP) [12,[39][40][41][42][43]. However, it is also demonstrated that SHP is able to upregulate gene transcription, as in the case of PPARα and PPARγ [44][45][46] and NF-κB [44].
Both N-terminal NR interaction domain and C-terminal domain of SHP are important for repression [47,48]. Overall, the SHP-mediated repression of target transcription factors occurs by at least three distinct transcriptional repression mechanisms ( Figure 2).
A first mechanism involves direct interference with the AF-2 coactivator domain of NRs (competition for coactivator binding, leading to the repression of NR-mediated transcriptional activity). This is the case for the inhibition of estrogen receptor α (ERα) and estrogen receptor β (ERβ) [49].
A third mechanism of inhibition of SHP involves the direct interaction with the surface of NR or transcription factor, resulting in the blockade of DNA binding and the consequent inhibition of its transcriptional activity. This is the case for RAR-RXR heterodimers [10], PXR-RXR binding to DNA by SHP [1], interaction with hepatocyte nuclear factor (HNF4), or Jun family of the activator protein 1 (AP-1) transcription factor complex (JunD) [51,52]. Genomic structure Figure 1: Top: the genomic structure of human SHP. Rectangles represent the two exons with a single intron spanning approximately 1.8 kilobases and located on a single locus on chromosome 1p36.1 [18]. The region 5 includes ≈600 nucleotides from the transcription start site and is characterized by promoter activity. Bottom: typical nuclear receptor is compared with the domain structure of human SHP. The canonical structure of NR includes the N-terminal activation function 1 (AF1) domain, DNA-binding domain (DBD), ligand-binding domain (LBD), and C-terminal activation function 2 (AF2) domain. SHP lacks the DBD. Two functional LXXLL-related motifs (also named as NR boxes) are typical of the human SHP structural domains. Such motifs are located in the putative N-terminal helix 1 of the LBD and in the C-terminal region of the helix 5. While active NRs exhibit glutamic acid in AF-2, the SHP AF-2 domain is replaced with aspartic acid. Adapted from Chanda et al. [15] and Shulman and Mangelsdorf [130].
All three mechanisms might occur sequentially or alternatively according to type of cells and promoters [12].
Clearly, information on factors that increase or decrease SHP expression and that are regulated by SHP is essential for understanding the regulatory effects of this orphan NR. Few years of research have not been enough to identify a true ligand. Interestingly, it is suggested that targeting posttranslational modifications of SHP may be an effective therapeutic strategy. Selected groups of genes could be controlled to cure a vast range of metabolic and SHP-related diseases [53]. Overall, the huge amount of information on SHP function is currently available, making this NR essential in a number of functions involving cholesterol and bile acid metabolism, lipogenesis, glucose metabolism, steroid hormone biosynthesis, xenobiotic homeostasis/metabolism, and cell cycle.
In particular, the ability of SHP in interacting with different metabolic signaling pathways including bile acids and lipid homeostasis, fat mass, adipocytes, and obesity will be reviewed here.

Bile Acids and Lipid Homeostasis
The wide ability of SHP to target multiple genes in diverse signaling pathways points to the key role of SHP in various biological processes, including the metabolism of bile salts, glucose, and fatty acids. Both unique structure and functional properties account for the complexity of SHP signaling. Studies suggest that loss of SHP might positively affect cholesterol and bile acid homeostasis in pathophysiologically relevant conditions [54]. Bile acids (BAs) are amphipatic cholesterol metabolites which are synthesized in the liver, secreted into bile, stored in the gallbladder, and secreted postprandially into the duodenum. BAs are synthesized from cholesterol, and this pathway provides the elimination of excess cholesterol in the body [55]. Moreover, BAs should be seen as physiological detergents which, in the small intestine, are essential for the absorption, transport, and distribution of lipophilic molecules, including dietary lipids, steroids, and lipid-soluble vitamins. In the intestine, BAs undergo extensive metabolism by the intestinal microflora. A high efficient system is the enterohepatic circulation of BAs [55,56], where more than 90-95% of BAs are returned to the liver from the terminal ileum via the portal vein. Thus, the concentration of BAs in serum, liver, and intestine is tightly regulated to prevent damage to enterohepatic tissues due to their strong detergent moiety [57][58][59]. The major rate-limiting step in biosynthetic pathway of BAs in humans is initiated by cholesterol 7α-hydroxylase (CYP7A1), the microsomal P450 liver enzyme, to produce two primary BAs, cholic acid, and chenodeoxycholic acid, essential in the overall balance of cholesterol homeostasis. Sterol 12α hydroxylase (CYP8B1) catalyzes the synthesis of cholic acid, a step which determines the cholic acid to CDCA ratio in the bile [60]. Secondary bile acids (deoxycholic acid and lithocholic acid) and tertiary bile acids (ursodeoxycholic acid) in humans are produced following intestinal dehydroxylation of primary bile acids by intestinal bacteria [58,61].
Regulation of BA biosynthesis is highly coordinated and is mediated by key NRs including the orphan receptor, liver receptor homologue-1 (LRH1; NR5A2), the hepatocyte nuclear factor 4α (HNF4α), SHP, and the bile acid receptor farnesoid X receptor (FXR; NR1H4). Thus, the activation of FXR initiates a feedback regulatory loop via induction of SHP, which suppresses LRH-1-and HNF4α-dependent  [134].

LXRα
Direct regulation of SHP and repression of CYP7A1-mediated bile acid biosynthesis (in humans not in rodents). Effect on cholesterol homeostasis [135].

PPARγ
Liver/PPARγ decreases gluconeogenic gene expression by the PPARγ/RXRα heterodimer binding to the PPRE in the human SHP promoter. A mechanism explaining the SHP-mediated acute antigluconeogenic effects of PPARγ [137].

SF-1
At least five binding sites for SF-1 detected in the promoter region of SHP. Rat testis and adrenal glands, human fetal adrenal gland [136]. Liver/SHP displays a circadian expression pattern involving CLOCK-BMAL1 (core circadian clock component). Regulation of SHP promoter together with LRH-1 and SHP. Relevance for circadian liver function? [38].

HNF4α
Pancreatic β-cells/decreased expression of SHP may be indirectly mediated by a downregulation of HNF4α. SHP can repress its own transcriptional activation by inhibiting HNF4 α function (feedback autoregulatory loop) and, indirectly (via HNF4 α), HNF1α function. Relevance for pancreatic islet differentiation, insulin secretion, synthesis [116]. JNK/c-Jun/AP-1 Primary rat hepatocytes/bile acid downregulation of CYP7A1-dependent bile acid biosynthesis via the JNK/cJun/AP1 pathway. SHP promoter is a direct target of activated c-Jun binding to AP-1 element [139]. Also, in HL-60 leukemia cells, c-Jun increases the transcriptional activation of the SHP promoter to activate the expression of Shp genes associated with the cascade regulation of monocytic differentiation [140]. SREBP-1 Liver/effect on human (not mouse) SHP promoter. Cholesterol and bile acid homeostasis, fatty acid synthesis [27].

USF-1
HepG2, H4IIE, and AML12 cells/HGF activates AMPK signaling pathway in hepatocytes, E-box-binding transcription factor USF-1, and binding to the Shp gene promoter. SHP induction of gene expression leads to inhibition of hepatic gluconeogenesis due to SHP-repressed transcription factor HNF4α [28].

SRC-1
Murine macrophage cell line RAW 264.7, HeLa, and CV-1 cells/SHP interacts negatively with SRC-1 (a transcription coactivator of nuclear receptors and other transcription factors including NF-κB). See also oxLDL in this table [44]. Active extract from Commiphora Mukul. FXR antagonist. In Fisher rats, guggulsterone increased transcription of bile salt export pump (BSEP) mRNA and SHP expression [29].

PGC-1α (gene expression inducer)
COS-7 cell lines/PGC-1α mediates the ligand-dependent activation of FXR and transcription of Shp gene. Relevance in mitochondrial oxidative metabolism in brown fat, skeletal muscle, and liver gluconeogenesis [33]. PMRT1 (group of protein arginine methyltransferases) Hepatic cell lines/PRMT1 functions as FXR coactivator and has a role in chromatin remodeling. PRMT1 induces BSEP and SHP and downregulation of NTCP and CYP7A1 (targets of SHP) [30].

oxLDL (oxidized low density lipoprotein)
Murine macrophage cell line RAW 264.7, HeLa, and CV-1 cells/oxLDL decreased SHP expression. SHP transcription coactivator of NF-κB which became progressively inert in oxLDL-treated RAW 264.7 cells (see also Table 3). Relevance for differentiation mechanism of resting macrophage cells into foam cells and resulting atherogenesis [44].
expression of the two major pathway enzymes cholesterol 7hydroxylase (CYP7A1) and sterol 12 hydroxylase (CYP8B1). The BA feedback regulation primarily occurs since BAs act as transcriptional regulators for the expression of the gene encoding CYP7A1. Both cholic acid and chenodeoxycholic acid function as endogenous ligands for the nuclear bile acid receptor FXR [62]. FXR expression is high in the intestine and liver, the two sites where BAs reach high concentrations to activate FXR. The transcription by FXR includes heterodimerization with retinoid X receptors (RXRs) in the cytoplasm, translocation into the nucleus, and binding to DNA response elements in the regulatory regions of target genes [63]. When the bind of BAs to FXR, SHP transcription is increased [60,64,65], this alteration leads to the inhibition of LRH-1 activity or HNF4α on the BA response elements (BAREs) of CYP7A1 and CYP8B1 promoters [64,65]. In this scenario, BA synthesis is downregulated by a precise feedback regulatory mechanism, which represents the major pathway under normal physiological conditions [64][65][66] (Figure 3). LRH1 is also a well-known activator of Shp gene transcription [64,65], and this step leads to an autoregulatory loop of gene expression by SHP [42]. This step also includes the G protein pathway suppressor 2 (GPS2) interacting with FXR, LRH-1, and HNF4α to regulate CYP7A1 and CYP8B1 expression in human hepatocytes [67] (Table 3). A critical role in maintaining cholesterol homeostasis for CYP7A1 has been recently advocated in a model of in Cyp7a1-tg mice [68].
The hepatocyte nuclear factor-1α (HNF1α), which haploinsufficiency causes the Maturity-onset diabetes of the young type 3 (MODY3), also appears to modulate SHP expression  [143]. Prevention of tamoxifen-induced estrogen agonistic effects and neoplastic changes in the endometrium in women with breast cancer taking tamoxifen?
ERRγ HeLa (human cervical carcinoma), CV-1 (green monkey kidney), and HEK 293 (human embryonic kidney) cell lines/SHP inhibits ERRγ transactivation by physical interaction with the 3 members of the ERR subfamily. Interaction is dependent on N-terminal receptor interaction domain of SHP and AF-2 surface of ERRγ. Part of the autoregulatory mechanism of gene expression going through ERRγ/SHP/ERRγ. A potential role in some forms of moderate human obesity during SHP mutations [133].
GR 293 human embryo kidney cells and COS-7 monkey kidney/SHP inhibits the transcriptional activity of GR via the LXXLL motif. Physiological role of SHP in glucocorticoid signaling and gluconeogenesis [22]. See also HNF4 [90] and Foxo1 [115].

HNF4
Human ANG transgenic mice and HepG2 cells treated with bile acids/evidence that bile acids negatively regulate the human ANG gene through the FXR/SHP-mediated process (inhibition of the binding of HNF4 to the ANG promoter) [90]. Mechanisms: SHP binds the AF-2 region and the N-terminal region of HNF4 and inhibits the binding of HNF4 to DNA. Also, modulation of HNF4 activity by SHP has important metabolic effects and interacts with the pathway of gluconeogenesis [47](see text and Foxo1) [115].

LRH-1
HepG2 cells/SHP interacts directly with the orphan receptor LRH-1 (AF-2 surface) and competes with other coactivators, leading to repression of LRH-1 transcriptional activity [48]. Demonstration that repression of CYP7A1 and bile acid synthesis requires coordinate interaction/transcription of FXR/LRH-1/SHP autoregulatory cascade, essential for maintenance of bile acid-induced negative feedback, and therefore hepatic cholesterol metabolism [65] (see also Figure 2).

Nur77 (NGFI-B)
HepG2 cells/Nur77 plays a key role in apoptosis of many cell types and cancer cells. Evidence that SHP functions to repress the transcriptional function of Nur77 (binding coactivator CBP, see elsewhere in this  table). SHP plays a protective role in the Nur77-mediated apoptosis in liver. Mutations in SHP: a role also for affect initiation and progression of inflammatory liver diseases such as alcoholic hepatitis and hepatic viral infections? [32].

PPARα
In vitro binding assays and in vivo experiments/the promoter regions of the genes encoding the first two enzymes of the peroxisomal beta-oxidation pathway (AOx, HD), contain transcriptional regulatory sequences (PPRE) bound by the PPARα/RXRα heterodimeric complex. SHP-inhibited transcription by PPARα/RXRα heterodimers from the AOx-PPRE. SHP potentiated transcription by PPARα/RXRα heterodimers from the HD-PPRE (evidence of SHP-dependent upregulation PPARα-mediated gene transcription) [46].

PPARγ
In vitro experiments, COS-7 cells/Shp gene expressed also in adipose tissue. SHP induces PPAR activation via C terminus (direct binding to the DBD/hinge region of PPARγ) and inhibition of the repressor activity of NCoR. SHP may act as an endogenous enhancer of PPARγ by competing with NCoR [45]. Mutant SHP proteins display less enhancing activity for PPARy compared with wild-type SHP, and a human model leading to mild obesity and insulin resistance has been described in Japanese during naturally occurring mutations [111] (see also text and Table 3).  [41]. Relevance for expression of several genes involved in drug and hormone metabolism [145].

BETA2/NeuroD
293T, COS-7, CV-1 cells/BETA2/NeuroD is a member of tissue-specific class B bHLH proteins and cats as a positive regulator of insulin gene expression [146] and neuronal differentiation [147]. SHP physically interacts and inhibits helix-loop-helix transcription factor BETA2/NeuroD transactivation of an E-box reporter in mouse pancreas islets. The inhibitory effect of SHP requires its C-terminal repression domain, interference with coactivator p300 for binding to BETA2/NeuroD, and direct transcriptional repression function. Relevance for development of the nervous system and the maintenance and formation of pancreatic and enteroendocrine cells [148].

C/EBPα
HepG2 hepatoma cells/SHP interacts directly with C/EBPα and represses C/EBPα-driven PEPCK gene transcription. Overall, a role for SHP in regulation of hepatic gluconeogenes is driven by C/EBPα activation in the liver [149].

Foxo1
C57BL/6J mice and HepG2 and HEK293T cells/treatment with chenodeoxycholic acid was associated with FXR-dependent SHP induction, downregulation of gluconeogenic gene expression (G6Pase, PEPCK, FBP1), interaction of the forkhead transcription factor Foxo1 with SHP, and repression of Foxo1-mediated G6Pase transcription (competition with CBP). A similar mechanism is postulated for SHP-driven HNF-4 repression of PEPCK, FBP1 transcription. A mechanism by which bile acids metabolism is linked to gluconeogenic gene expression via an SHP-dependent regulatory pathway [115].
Jun D Two rat models of liver fibrosis and Hepatic Stellate cells (HSC)/promoting the ligand-induced FXR-SHP cascade (by the FXR ligand 6-EDCA, in rat models) and overexpressing SHP in HSC prevented fibrogenic changes in the liver. SHP binds JunD and inhibits DNA binding of adaptor protein (AP)-1 induced by thrombin. FXR ligands as therapeutic agents to treat liver fibrosis? [52].

NF-κB
Murine macrophage cell line RAW 264.7/SHP acts as a positive transcription coactivator of NF-κB and essential for NF-κB transactivation by palmitoyl lysophosphatidylcholine (one of the oxLDL constituents). Relevance for differentiation mechanism of resting macrophage cells into foam cells and resulting atherogenesis (see also [44]).

TRAF6, p65
Macrophages/a novel function of SHP in innate immunity involving Toll-like receptors (TLRs). SHP negatively regulates TLR signaling to NF-κB. Likely, SHP negatively regulates immune responses initiated by various pathogen-recognition receptors by forming a complex with TRAF6 and effect on TRAF6 ubiquitination. In the cytosol of LPS-stimulated cells. SHP also acts as specific transrepressor of the transcription factor p65 (part of the p50/p65 heterodimer found in NF-κB). An additional role for SHP in sepsis and inflammatory disease? [128,129]. 8 HepG2 cells/The CYP7A1 gene was used as a model system. SHP has direct interaction with corepressors at the level of native chromatin. SHP directly interacted and mediated the recruitment of mSin3A-Swi/Snf-Brm chromatin remodelling complex to the CYP7A1 promoter (TATA and BARE II region of the promoter). Also, the mSinA3/HDAC1 corepressor complex is inhibiting transcription by histone deacetylation. SHP also interacted with known proteins belonging to the Swi/Snf complex (BAF155, BAF47). This mechanism explains the complex and subtle SHP-driven inhibition of hepatic bile acid synthesis [50].

CBP
HepG2 cells, CV-1 cells/SHP binds coactivator CBP and competes with Nur77. The mechanism explains the repression of the transcriptional function of Nur77, which is fundamental in apoptosis in the liver [32].

SIRT1
HepG2, HEK293T (293T), and HeLa cells/SIRT1 is a HDAC of class III. SHP recruits SIRT1 (activating deacetylase activity of SIRT1) to repress LRH1 transcriptional activity as well as inhibition LRH1 target gene promoter activity and mRNA levels. A novel mechanism is described for SHP repressive action and control of bile acid homeostasis. SIRT1 in working concertedly with NRs and affecting chromatin remodeling in target gene promoters [42].

SMRT/NcoR
Hepatoma cell lines/studies on the role of SHP in CAR-mediated transactivation of the CYP2B gene. SHP might interact with subunits of functionally distinct coregulator complexes, including HDAC3-N-CoR-SMRT [24,120]. SHP −/− mice/SHP as an important transcriptional activator of miRNA-206 gene expression via a cascade dual inhibitory mechanism involving AP1 but also YY1 and ERRγ. Relevance for multiple steps involving cellular development, proliferation, and differentiation [153].

RNA Pol II
Caco-2 cells/within the pathway of SHP-LXR interaction, it is shown that SHP can interact in vitro with RNA polymerase II but not with TFIID and TFIIE transcription initiation factor II D (TFIID), general transcription factor II E (TFIIE) (components of the basal transcription machinery). A further mechanism by which SHP could inhibit both basal and induced transactivation [144]. via the FXR pathway. In this respect, HNF1α (−/−) mice displayed a defect in bile acid transport, increased bile acid and liver cholesterol synthesis, and impaired HDL metabolism [69].
A role for SHP in mediating the recruitment of mSin3A-Swi/Snf to the CYP7A1 promoter, with chromatin remodeling and gene repression, has been described. In HepG2 cells, Kemper et al. [50] have shown that bile acid treatment resulted in SHP-mediated recruitment of transcriptional coregulators mSin3A and Swi/Snf complex to the promoter, chromatin remodeling, and gene repression (Table 3). This is an additional mechanism involving transformation of nucleosome conformation for the repression by SHP of genes activated by various NRs. In line with such results, increased synthesis and accumulation of BAs occurs in SHP (−/−) mice, due to the loss of SHP repression and consequent derepression of the rate-limiting CYP7A1 and cholesterol 12αhydroxylase (CYP8B1) (the rate-determining enzyme of the alternative but minor BA synthesis pathway) in the biosynthetic pathway [70][71][72].
Another study demonstrated, in SHP (−/−) mice on a background of 129 strain, the protection against hypercholesterolemia in three different models: an atherogenic diet, hypothyroidism, and SHP (−/−) mice intercrossed with LDLR (−/−) mice (to generate SHP/LDLR double (−/−) mice in a mixed 129-C57BL/6 background). When fed an atherogenic diet, the latter strain was almost completely resistant to diet-mediated increases in triglyceride, very lowdensity lipoprotein (VLDL) cholesterol, and low-density lipoprotein (LDL) cholesterol but had an increase in highdensity lipoprotein (HDL) cholesterol as compared with LDLR (−/−) mice. Such results point to the protection against dyslipidemia following the inhibition of hepatic SHP expression, although no antagonist ligands have yet been identified for SHP [78]. We have recently examined biliary lipid secretion and cholesterol gallstone formation in male SHP (−/−) and (+/+) mice before and during the feeding of a lithogenic diet for 56 days [79]. Deletion of the Shp gene significantly increased hepatic bile salt synthesis, and doubled the increase of biliary bile salt outputs in SHP (−/−) mice than in (+/+) mice. The intestinal bile acid pool size was significantly greater in SHP (−/−) mice than in (+/+) mice. These increased BAs are efficacious ligands of FXR and can stimulate the expression of intestinal fibroblast growth factor 15 (FGF15) in mice through the FXR signaling pathway, which is consistent with the expanded bile acid pool size in SHP (−/−) mice. At 14 days on the lithogenic diet, fasting gallbladder volume was significantly larger in SHP (+/+) mice than in (−/−) mice [80].
Indeed, FGF15/19 (mouse and human orthologs, resp.) is another FXR gene target in the intestine and appears to contribute to the fine tuning of bile acid synthesis in the liver. Thus, a model for FXR-mediated repression of bile acid synthesis should also take into account the bile acid-mediated activation of intestinal FXR and FGF15 in the small intestine (while the FXR-SHP pathway is activated in the liver). According to the most plausible view, FGF15 acts as a hormone to signal between intestine and liver. The secreted FGF15 by the intestine circulates to the liver, likely through The potential molecular mechanisms of crosstalk between nuclear receptors LXR and FXR-SHP-LRH-1 regulatory cascade in the liver and intestine. Bile acids act as ligands for FXR, which regulates transcription by binding as a heterodimer with RXRs. This step results in increased SHP expression. SHP in turn inhibits LRH-1, preventing the activation of target genes that participate in bile acid and fatty acid synthesis. In the absence of bile acids, LRH-1 acts together with LXR to stimulate bile acid synthesis [64,65,132]. The important pathways in the intestine that contribute to modulation of bile acid synthesis are also depicted (see text for details). There is a bile-acid-mediated activation of intestinal FXR and, as a result, the release of FGF15 in the small intestine. The secreted FGF15 by the intestine circulates to the liver, likely through the portal circulation or lymph flow [81] and induces the activation of FGFR4 in the liver. The FGF15/FGFR4 pathway synergizes with SHP in vivo to repress CYP7A1 expression [57]. Bas: bile acids; FGF: fibroblast growth factor; FGFR4: FGF receptor; FXR: farnesoid X receptor; LRH-1: liver receptor homologue-1; LXR: liver X receptor; RXR: retinoid X receptors; SHP: short heterodimer partner. Adapted from Ory [66] and Inagaki et al. [57]. the portal circulation or lymph flow [81], and induces the activation of FGFR4 in the liver. As shown in Figure 3, the FGF15/FGFR4 pathway synergizes with SHP in vivo to repress CYP7A1 expression [57]. In humans, a similar mechanism should involve the FGF19. Of note, activation of FXR transcription in the intestine protected the liver from cholestasis in mice by inducing FGF15 expression and reducing the hepatic pool of BA. This suggests a potential approach to reverse cholestasis in patients [82]. Hepatic fatty acid homeostasis is also regulated by SHP since regulating these genes involves in fatty acid uptake, synthesis, and export [83][84][85][86][87]. In a study exploring global gene expression profiling combined with chromatin immunoprecipitation assays in transgenic mice constitutively expressing SHP in the liver, overexpression of SHP in the liver was associated with the depletion of the hepatic bile acid pool and a concomitant accumulation of triglycerides in the liver [84]. By contrast, fat accumulation induced by a high-cholesterol or high-fat diet is prevented by the deletion of SHP [88,89]. The pleiotropic role of SHP can also be found in the case of nonalcoholic liver steatosis since OB/SHP double (−/−) mice (a model of severe obesity and insulin resistance) became resistant to liver steatosis and showed improved insulin sensitivity [86].
Another interesting role for SHP emerged after it was found that BAs negatively regulate the human angiotensinogen (ANG) gene. ANG is the precursor of vasoactive octapeptide angiotensin II, and BAs act through the SHP pathway by preventing hepatocyte nuclear factor-4 (HNF4) from binding to the human ANG promoter [90].

Fat Mass, Adipocytes, and Obesity
SHP appears to play a central role in obesity. Human obesity is considered a polygenic disorder characterized by partly known abnormal molecular mechanisms resulting in increased fat mass, with an imbalance between the energy acquired from nutrients that dissipated as heat (i.e., thermogenesis). In this respect, weight stability requires a balance between calories consumed and calories expended [91]. In adipose tissue depots, two main types of adipocytes exist, that is, brown adipocytes and white adipocytes. In several animal species, some adipose tissue sites mainly include brown adipocytes (BATs) and the other contains mainly white adipocytes (WATs). BAT dissipates chemical energy to produce heat either as a defense against cold [92] or as energy expenditure to compensate food intake [93,94]. The unusual function of BAT might be better understood by considering that they share a common origin with myocytes [95,96], and BAT was indeed considered something in between muscle and adipose tissue [95]. BAT is deemed as the major site for sympathetic (adrenergic) mediated adaptive thermogenesis; this pathway involves the uncoupling protein-1 (UCP1). WAT is mainly implicated in the regulation of lipid storage and catabolism but also in the synthesis and secretion of adipokines [97][98][99][100]. While the percentage of young men with BAT is high, the activity of BAT is reduced in men who are overweight or obese [101]. Thermogenesis unequivocally exists in both humans and animals, and BAT is the major site of thermogenesis which can be increased by environmental factors (i.e., adaptive thermogenesis). In both human and animal species, dietary composition, chronic cold exposure, and exercise may increase thermogenesis [102]. As far as adipose tissue biology is concerned, SHP seems to play a distinct regulatory function in WAT, as compared with BAT. A number of experiments have focused on animal models of obesity and subtle molecular changes. SHP-deficient mice are protected against high-fat-diet-induced obesity [89].
Peroxisome proliferator-activated receptor (PPAR) γ coactivator-1 (PGC-1) family members are multifunctional transcriptional coregulators. PGC-1 acts as a molecular switch in several metabolic pathways. In particular, PGC-1α and PGC-1β regulate mitochondrial biogenesis, adaptive thermogenesis, fatty acid and glucose metabolism, fiber-type switching in skeletal muscle, peripheral circadian clock, and development of the heart [103]. In particular, SHP functions as a negative regulator of energy production in BAT [89] because SHP is a negative regulator of PGC-1α expression in BAT. In turn, PCG-1α is a coactivator of uncoupling protein 1 (UCP1) which plays a major role in energy dissipation as heat in multilocular BAT of different animal species and humans [104][105][106]. Fat-specific (BAT) SHP-overexpressed transgenic mice had increased body weight and adiposity. Energy metabolism, however, was increased, and BAT cold exposure function was enhanced with activation of thermogenic genes and mitochondrial biogenesis (enhanced β1-AR gene expression and PGC1α). Compared with wild-type mice on a high-fat diet, SHP overexpression was associated with enhanced diet-induced obesity phenotype with weight gain, increased adiposity, and severe glucose intolerance. An additional feature of SHP transgenic mice was a decreased diet-induced adaptive thermogenesis, increased intake of food, and decreased physical activity [107]. This leads to the conclusion that, although expressed at low levels in fat, activation of SHP in adipocytes has a strong effect on weight gain and diet-induced obesity [107]. Moreover, if mechanisms linked to energy metabolism and the development of obesity are considered, SHP has distinct roles in WAT and BAT. As previously mentioned, while SHP deletion in obese leptin-deficient mice (ob/ob) prevented the development of nonalcoholic fatty liver and improved peripheral insulin sensitivity [86], SHP deletion did not overcome the severe obesity caused by leptin deficiency. A significant protective effect from obesity by SHP deficiency was likely associated with the low basal level of SHP expressed in fat. Adipogenesis appears to be influenced by SHP: when SHP was overexpressed in 3T3-L1 preadipocytes, cell differentiation was inhibited, as well as the accumulation of neutral lipids within the cells. Thus, SHP may act as a molecular switch governing adipogenesis. In particular, SHP appears to be a potent adipogenic suppressor, and preadipocytes are kept in an undifferentiated state through the inhibition of the adipogenic transcription factors and stimulators [108]. Further studies will address whether the loss of SHP function results in inhibition of lipid accumulation in adipocytes, similar to what is observed in hepatocytes. In a future clinical setting, treatment of obesity might also include drugs able to mimic or stimulate the effects of SHP. Mutations in the Shp gene have also been reported in patients with lipodystrophy carrying four different polymorphisms [109].
SHP mutations may not be considered a common cause of severe obesity. A number of important clinical studies have examined this issue (Table 4); however, Hung et al. [110] in UK examined the relationships between genetic variation in SHP and weight at birth, adiposity, and insulin levels in three different populations (the Genetics of Obesity Study) GOOS, the Avon Longitudinal Study of Parents and Children (ALSPAC), and the Ely studies). In the 329 cases of severe early-onset obesity (GOOS study), two novel and rare missense mutations (R34G and R36G) were identified which might in part contribute to obesity in the probands. Furthermore, two common polymorphisms, namely, G171A (12% of subjects with higher birth weight) and −195CTGAdel (16% of subjects with lower birth weight) were found. In the ALSPAC cohort of 1,079 children, the G171A variant was associated with increased body mass index and waist circumference together with higher insulin secretion 30 minutes after glucose load. Thus, whereas mutations in the Shp gene cannot be seen as a common cause of severe human obesity, genetic variation in the Shp gene locus may influence birth weight and have effects on body size. The effect might ultimately involve insulin secretion by the negative regulation between SHP and the hepatocyte nuclear factor-4α (HFN-4α), a transcription factor involved in differentiation and function of pancreatic β-cells [110].
A possibility is that decreased SHP expression or function results in increased HFN-4α activity with a cascade of events, including fetal hyperinsulinemia, and increased birth weight. At a later stage, sustained hyperinsulinemia might be responsible of insulin resistance and obesity of the adult [110].
Mutations in the Shp gene were also associated with influence on birth weight, mild obesity, and insulin levels in the study by Nishigori et al. on 274 Japanese subjects [111]. Mutations in several genes encoding transcription factors of the hepatocyte nuclear factor (HNF) cascade are associated with maturity-onset diabetes of the young (MODY). MODY is a monogenic form of early-onset diabetes mellitus (defective insulin secretion with normal body weight), and SHP is deemed as a plausible candidate MODY gene; this is because SHP is able to inhibit the transcriptional activity of the hepatocyte nuclear factor-4α (HFN-4α), a key member of the MODY regulatory network. Thus, further studies have looked for segregation of SHP mutations with MODY in a cohort of Japanese patients with early-onset diabetes. In this context, variants in SHP appeared to cosegregate with  increased body mass index in families, thus contributing to obesity among Japanese subjects. Also, increased risk of morbidity was observed in another study from Japan, examining patients with type 2 diabetes and SHP mutations [112]. Major differences, however, might exist in the prevalence and function of SHP variants in different populations. Of note, the results from other Caucasian cohorts did not confirm the association between SHP mutation and obesity [113,114]. Echwald et al. conducted an elegant study on the prevalence of SHP variants by single-strand conformational polymorphism and heteroduplex analysis among 750 Danish obese men with early-onset obesity [114]. As control, a cohort of 795 nonobese control subjects was genotyped using PCR-RFLP.  [111] c.512G>C [p.G171A] common polymorphism was identified; however, the prevalence of functional SHP variants associated with obesity was considerably lower among Danish subjects (1 out of 750 obese, none of control subjects), compared to the prevalence observed in Japan by Nishigori et al. [111]. Mitchell at al. [113] investigated SHP variants in 1927 UK subjects according to type 2 diabetes, obesity, and birth weight. Although reporting a raised body mass index among homozygous carriers of the 171A variant (<1%), this polymorphism was unlikely to be associated with all three conditions in Caucasians. Taken together, the above-mentioned studies suggest that the 171A variant might contribute only to subsets of polygenic obesity.

Other Functions of SHP
The existence of multiple interactions of SHP with NRs, transcription factors and transcriptional cofactors (Tables 2  and 3) points to the pleiotropic and central role of SHP in the body.
SHP has been hypothesized to act in glucose homeostasis via complex pathways involving the inhibition of glucocorticoid receptors (GR) in mammalian cells and the inhibition of PGC-1 gene, a coactivator of NRs important for gluconeogenic gene expression and the PGC-1-regulated phospho(enol)pyruvate carboxykinase (PEPCK) promoter. Such steps underscore a physiologically relevant role for SHP in modulating hepatic glucocorticoid action [22]. Following the bile acid-induced induction, SHP inhibited a number of other pathways, including the HNF4α-mediated transactivation of the PEPCK and fructose biphosphate (FBP) promoters, as well as the transactivation of the glucose-6phosphatase (G6Pase) promoter mediated by Foxo1 [115]. The interaction between SHP inhibitory function and the 3 isoforms (α, β, and γ) of the hepatocyte nuclear factor-3 (HNF4) points to the regulatory role of SHP on gluconeogenesis [51]. A role for SHP in insulin secretion pathway has also been reported. Mutations in hepatocyte nuclear factor 1α (HNF-1α) is associated with maturity-onset diabetes of the young type 3. This condition depends on impaired insulin secretory response in pancreatic beta cells.
Indeed, loss of HNF-1α function in HNF-1α (−/−) mice resulted in altered expression of genes involved in glucosestimulated insulin secretion, but also insulin synthesis, and beta-cell differentiation. Pancreatic islets of HNF-1α (−/−) mice showed a distinctive reduction of SHP expression and a downregulation of the HNF4α gene expression. Since SHP appears to repress its own transcriptional activation following heterodimerization with HNF4α, a feedback autoregulatory loop between SHP and HNF4α has been hypothesized [116]. Also, SHP likely functions as a negative regulator of pancreatic islet insulin secretion. SHP (−/−) mice were characterized by hypoinsulinemia, increased glucosedependent response of islets, increased peripheral insulin sensitivity, and increased glycogen stores [117]. The role played by SHP in the regulation of hepatic gluconeogenesis has also emerged in a number of additional experiments. For example, the liver of SHP (−/−) mice showed increased glycogen stores [117], while hepatic Shp gene expression (induced by the antidiabetic biguanide drug metformin) was associated with inhibition of hepatic gluconeogenesis. Induction of SHP was achieved via AMP-activated protein kinase (AMPK) and associated with downregulation of essential gluconeogenic enzyme genes, that is, phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase) [118], and fructose-1,6-bisphosphatase (FBP1) [119].
PGC-1 gene is a coactivator of NRs, and this step is relevant for gluconeogenic gene expression. Yamagata et al. [119] showed that bile acid (chenodeoxycholic acid) was able to induce the downregulation of PGC-1 gene, and this mechanism involved forkhead transcription factors (Foxo1, Foxo3a, Foxo4) via a SHP-dependent manner.
Drug metabolism and detoxification might be regulated by SHP. This is also the case for excess BAs: the pregnane X receptor (PXR) induces CYP3A and inhibits CYP7α, both involved in biochemical pathways leading to the conversion of cholesterol into primary BAs, whereas CYP3A is also involved in the detoxification of toxic secondary bile acid derivatives. SHP acts as a potent repressor of PXR transactivation, and this finding suggests that PXR can act on both bile acid synthesis and elimination detoxification [1]. Additional mechanisms involved in the SHP-dependent control of pathways of drug metabolism have been identified. The expression of genes involved with the metabolism of xenobiotics might be regulated by SHP in the spleen acting on (aryl hydrocarbon receptor (AHR)/AHR nuclear translocator (ARNT)) AHR/ARNT heterodimers which, in turn, bind to xenobiotic response elements (XREs) at the level of specific DNA sequences [41]. A number of genes involved in hormone and drug metabolism would be expressed (i.e., UGT16, ALDH3, CYP1A1, CYP1A2, CYP1B1, etc.). SHP also appears to downregulate the constitutive-androstanereceptor-(CAR-) mediated CYP2B1 gene expression, induced by phenobarbital to form the CAR/RXR heterodimer which, in turn, binds to 2 DR-4 sites to form the phenobarbital responsive unit in the CYP2B gene [120] (Table 3). One role of SHP in steroidogenesis has been identified in the testes  with influence on testosterone synthesis and germ cell differentiation [121] and in the intestine for glucocorticoid synthesis [122].
A role for SHP in cell proliferation and apoptosis signaling is emerging. Depending on the cell type, SHP seems to have both inhibitory and stimulatory effects on apoptosis. However, the manipulation of SHP through the synthetic ligands adamantyl-substituted retinoid-related (ARR) compounds 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthale-necarboxylic acid (CD437/AHPN) and 4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC) induces apoptosis of a number of malignant cells (i.e., leukemia and breast carcinoma) both in vitro and in vivo [123,124]. The complex mechanism implies binding of ARR and 3-Cl-AHPC to SHP with formation of a corepressor complex containing Sin3A and nuclear receptor corepressor (N-CoR) which activate local control of mitochondrial function and apoptosis, with a limiting function on tumorigenesis [17,123] (Table 3). SHP appears to be also involved in DNA methylation and acting as a tumor suppressor, at least in the human and mouse livers [125][126][127]. Whether manipulation of SHP will be helpful in the treatment of hepatic and other gastrointestinal cancers is still a matter of research. The recent finding that SHP negatively regulates TLR signaling to NF-κB has raised the interest for the role of SHP in mechanisms governing innate immunity. SHP appears to negatively regulate the expression of genes encoding inflammatory molecules. Of note, direct binding of NF-κB seems to occur in resting cells, while binding of SHP to TRAF6 occurs in LPS-stimulated cells [128,129].

Conclusions and Perspectives
A remarkable number of metabolic functions in the body appear to be regulated by the orphan unique NR, small heterodimer partner SHP, which targets a complex set of genes in multiple pathways as a transcriptional corepressor (Figure 4). Pathways include fatty acid metabolism, glucose homeostasis, and drug-hormone detoxification. When looking at complex mechanisms leading to some important lipidopathies, that is, obesity and liver steatosis, enlightening data about the regulatory function of SHP are provided by studies using Shp-deleted and Shp-overexpressed animal models. Most likely, a condition of Shp deficiency might counteract lipid accumulation and improve plasma lipoprotein profiles. Further studies are urgently needed to confirm that such an important metabolic regulatory mechanism of SHP is true and has high translational value. To date, however, no synthetic antagonists or agonists for SHP are available, and one should keep in mind that rather divergent and somewhat elusive data have been observed regarding the loss of SHP function in humans and rodents. Thus, careful examination of subtle SHP intrinsic functions is essential to dissect potential modulatory pathways of SHP for a variety of metabolic abnormalities but also in tumorigenesis. Moreover, identifying specific endogenous ligands and synthetic agonists of SHP will pave the way to for therapeutic intervention. The effect of synthetic ligands on SHP modulation in hepatocytes and adipocytes, for example, might represent therapeutic tools for the treatment of constituents of the metabolic syndrome, namely, hypercholesterolemia, overweight obesity, and liver steatosis.