SHP mRNA Research Articles

Hepatocyte-specific deletion of small heterodimer partner protects mice against acetaminophen-induced hepatotoxicity via activation of Nrf2.

Acetaminophen (APAP) overdose stands as the primary cause of acute liver failure in the United States. APAP hepatotoxicity involves hepatic glutathione (GSH) depletion and mitochondrial damage. To counteract the toxicity of APAP, the nuclear factor erythroid 2 like 2 (Nrf2) activates the expression of genes responsible for drug detoxification and GSH synthesis. In this study, we present evidence that the elimination of hepatocyte small heterodimer partner, a critical transcriptional repressor for liver metabolism, results in Nrf2 activation and protects mice from APAP-induced acute liver injury. Initial investigations conducted on wildtype (WT) mice revealed a swift downregulation of Shp mRNA within the first 24 h after APAP administration. Subsequent treatment of hepatocyte-specific Shp knockout (ShpHep-/-) mice with 300 mg/kg APAP for 2 h exhibited comparable bioactivation of APAP with that observed in the WT controls. However, a significant reduction in liver injury was observed in ShpHep-/- after APAP treatment for 6 and 24 h. The decreased liver injury correlated with a faster recovery of GSH, attributable to heightened expression of Nrf2 target genes involved in APAP detoxification and GSH synthesis. Moreover, in vitro studies revealed that SHP protein interacted with NRF2 protein, inhibiting the transcription of Nrf2 target genes. These findings hold relevance for humans, as overexpression of SHP hindered APAP-induced NRF2 activation in primary human hepatocytes. In conclusion, our studies have unveiled a novel regulatory axis involving SHP and NRF2 in APAP-induced acute liver injury, emphasizing SHP as a promising therapeutic target in APAP overdose-induced hepatotoxicity.

Small Heterodimer Partner Modulates Macrophage Differentiation during Innate Immune Response through the Regulation of Peroxisome Proliferator Activated Receptor Gamma, Mitogen-Activated Protein Kinase, and Nuclear Factor Kappa B Pathways.

Hepatic macrophages act as the liver's first line of defense against injury. Their differentiation into proinflammatory or anti-inflammatory subpopulations is a critical event that maintains a delicate balance between liver injury and repair. In our investigation, we explored the influence of the small heterodimer partner (SHP), a nuclear receptor primarily associated with metabolism, on macrophage differentiation during the innate immune response. During macrophage differentiation, we observed significant alterations in Shp mRNA expression. Deletion of Shp promoted M1 differentiation while interfering with M2 polarization. Conversely, overexpression of SHP resulted in increased expression of peroxisome proliferator activated receptor gamma (Pparg), a master regulator of anti-inflammatory macrophage differentiation, thereby inhibiting M1 differentiation. Upon lipopolysaccharide (LPS) injection, there was a notable increase in the proinflammatory M1-like macrophages, accompanied by exacerbated infiltration of monocyte-derived macrophages (MDMs) into the livers of Shp myeloid cell specific knockout (Shp-MKO). Concurrently, we observed significant induction of tumor necrosis factor alpha (Tnfa) and chemokine (C-C motif) ligand 2 (Ccl2) expression in LPS-treated Shp-MKO livers. Additionally, the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways were activated in LPS-treated Shp-MKO livers. Consistently, both pathways were hindered in SHP overexpression macrophages. Finally, we demonstrated that SHP interacts with p65, thereby influencing macrophage immune repones. In summary, our study uncovered a previously unrecognized role of SHP in promoting anti-inflammatory macrophage differentiation during the innate immune response. This was achieved by SHP acting as a regulator for the Pparg, MAPK, and NF-κB pathways.

Liver-Specific Bmal1 Depletion Reverses the Beneficial Effects of Nobiletin on Liver Cholesterol Homeostasis in Mice Fed with High-Fat Diet.

Nobiletin (NOB), a naturally occurring small-molecule compound abundant in citrus peels, has displayed potential lipid-lowering and circadian-enhancing properties in preclinical studies. However, the requirement of specific clock genes for the beneficial effects of NOB is not well understood. In the current study, mice with a liver-specific deletion of the core clock component, Bmal1-Bmal1LKO-were fed a high-fat diet (HFD) ad libitum for eight weeks, while NOB (200 mg/kg) was administered by daily oral gavage from the fifth week and throughout the last four weeks. NOB decreased liver triglyceride (TG) alongside the decreasing mRNA levels of de novo lipogenesis (DNL) genes in both Bmal1flox/flox and Bmal1LKO mice. NOB increased serum very low-density lipoprotein (VLDL) levels in Bmal1LKO mice, which was consistent with higher liver Shp and lower Mttp mRNA expression levels, the key genes that facilitate VLDL assembly and secretion. NOB decreased liver and serum cholesterol levels in the Bmal1flox/flox mice, consistent with lower Hmgcr and higher Cyp7a1, Cyp8b1, Gata4 and Abcg5 mRNA levels in the liver. In contrast, in the Bmal1LKO mice, NOB increased Hmgcr mRNA levels and had no effect on the above-mentioned genes related to bile acid synthesis and cholesterol excretion, which might contribute to the elevation of liver and serum cholesterol levels in NOB-treated Bmal1LKO mice. NOB inhibited hepatic DNL and decreased liver TG levels in HFD-fed mice independently of liver Bmal1, whereas liver-specific Bmal1 depletion reversed the beneficial effects of NOB on liver cholesterol homeostasis. The complex interactions between NOB, the circadian clock and lipid metabolism in the liver warrant further research.

Lactobacillus fermentum MCC2760 abrogate high-fat induced perturbations in the enterohepatic circulation of bile acids in rats

This study in hyperlipidemic rats elucidated the effect of Lactobacillus fermentum MCC2760 on intestinal bile acid (BA) uptake, hepatic BA synthesis, and enterohepatic BA transporters. Diets rich in saturated fatty acids [coconut oil (CO)] and omega-6 fatty acids [sunflower oil (SFO)] at 25g fat/100g diet were fed to rats with or without MCC2760 (109 cells/kg body weight). After 60days of feeding, intestinal BA uptake and expression of Asbt, Osta/b mRNA and protein, and hepatic expression of Ntcp, Bsep, Cyp7a1, Fxr, Shp, Lrh-1, and Hnf4a mRNA were measured. Hepatic expression of HMG-CoA reductase protein and its activity and total BAs in serum, liver, and feces were assessed. Hyperlipidaemic groups (HF-CO and HF-SFO) had: 1) increased intestinal BA uptake, Asbt and Osta/b mRNA expression, and ASBT staining 2) increased BA in serum, 3) decreased hepatic expression of Ntcp, Bsep, and Cyp7a1 mRNA, and NTCP staining 4) increased activity of HMG-CoA reductase, 5) increased hepatic expression of Fxr and Shp mRNA, 6) decreased hepatic expression of Lrh-1 and Hnf4a mRNA, and 7) decreased BA in Feces when compared to their respective controls (N-CO and N-SFO) and experimental groups (HF-CO+LF and HF-SFO+LF). Immunostaining revealed increased intestinal Asbt and hepatic Ntcp protein expression in the HF-CO and HF-SFO groups compared to control and experimental groups. Incorporating probiotics like MCC2760 abrogated hyperlipidemia-induced changes in the intestinal uptake, hepatic synthesis, and enterohepatic transporters of BA in rats. Probiotic MCC2760 can be used to modulate lipid metabolism in high-fat-induced hyperlipidemic conditions.

Sodium butyrate alleviates cholesterol gallstones by regulating bile acid metabolism

Cholesterol overloading and bile acid metabolic disorders play an important role in the onset of cholesterol gallstone (CGS). Short-chain fatty acids (SCFAs) can regulate bile acid metabolism by modulating the gut microbiota. However, the role and mechanism by which sodium butyrate (NaB) targets bile acids to attenuate CGS are still unknown. In this study, continuous administration of 12 mg/day for 8 weeks was decreased the incidence of gallstones induced by lithogenic diet (LD) from 100% to 25%. NaB modulated SCFAs and improved the gut microbiota. The remodeling of the gut microbiota changed the bile acid compositions and decreased cecal tauro-α-muricholic acid (T-α-MCA) and tauro-β-muricholic acid (T-β-MCA) which are effective farnesoid X receptor (FXR) antagonists. The quantitative real-time PCR examination showed that NaB significantly increased levels of ileal Fxr, fibroblast growth factor-15 (Fgf-15) and small heterodimer partner (Shp) mRNA and subsequently inhibited bile acid synthesis. In addition, NaB enhanced bile acid excretion by increasing the levels of hepatic multidrug resistance protein 2 (Mdr2) and bile salt export pump (Bsep) mRNA, and it enhanced bile acid reabsorption in the intestine by increasing the levels of ileal bile acid transporter (Ibat) mRNA. In addition, NaB reduced the absorption of cholesterol in the intestine and inhibited the excretion of cholesterol in the liver, which reduced the cholesterol concentration in serum and bile. Furthermore, the protective effects of NaB administration were abolished by FXR antagonists.Taken together, our results suggest that NaB mitigates CGS by modulating the gut microbiota to regulate the FXR-FGF-15/SHP signaling pathway.

Gastric Bypass Increases Circulating Bile Acids and Activates Hepatic Farnesoid X Receptor (FXR) but Requires Intact Peroxisome Proliferator Activator Receptor Alpha (PPARα) Signaling to Significantly Reduce Liver Fat Content

BackgroundWe interrogate effects of gastric bypass (RYGB), compared with a low-calorie diet, on bile acid (BA), liver fat, and FXR, PPARα, and targets in rats with obesity and non-alcoholic fatty liver disease (NAFLD). MethodsMale Wistar rats received a high-fat diet (obese/NAFLD, n=24) or standard chow (lean, n=8) for 12 weeks. Obese/NAFLD rats had RYGB (n=11), sham operation pair-fed to RYGB (pair-fed sham, n=8), or sham operation (sham, n=5). Lean rats had sham operation (lean sham, n=8). Post-operatively, five RYGB rats received PPARα antagonist GW6417. Sacrifice occurred at 7 weeks. We measured weight changes, fasting total plasma BA, and liver % steatosis, triglycerides, and mRNA expression of the nuclear receptors FXR, PPARα, and their targets SHP and CPT-I. ResultsAt sacrifice, obese sham was heavier (p<0.01) than all other groups that had lost similar weight loss. Obese sham had lower BA levels and lower hepatic FXR, SHP, and CPT-I mRNA expression than lean sham (P<0.05, for all comparisons). RYGB had increased BA levels compared with obese and pair-fed sham (P<0.05, for both), while pair-fed sham had BA levels, similar to obese sham. Compared with pair-fed sham, RYGB animals had increased liver FXR and PPARα expression and signaling (P<0.05). Percentage of steatosis was lower in RYGB and lean sham, relative to obese and pair-fed sham (P<0.05, for all comparisons). PPARα inhibition after RYGB resulted in similar weight loss but higher liver triglyceride content (P=0.01) compared with RYGB alone. ConclusionsRYGB led to greater liver fat loss than low-calorie diet, an effect associated to increased fasting BA levels and increased expression of modulators of liver fat oxidation, FXR, and PPARα. However, intact PPARα signaling was necessary for resolution of NAFLD after RYGB.

Pyridoxal isonicotinoyl hydrazone inhibition of FXR is involved in the pathogenesis of isoniazid-induced liver injury

Isoniazid (INH)-induced liver injury may be associated with inhibition of the liver farnesoid X receptor (FXR). However, the relationship between FXR and INH-induced liver injury remained unclear. The present study was performed to clarify the role of inhibition of FXR in the pathogenesis of INH-induced liver injury and to further identify potential inhibitors of FXR from INH and its metabolites. HepaRG cells were treated with INH (10 mM) plus mixed bile acids (BA) and rats were treated with INH (60–600 mg/kg p.o.) or INH plus obeticholic acid (OCA, 10 mg/kg), a potent FXR agonist, for seven days. INH can cause BA-dependent toxicity and apoptosis with elevated intracellular bile acids in vitro; indeed, in these studies, liver bile acids and mRNA levels for Cyp7a1, an FXR target gene were increased, while mRNA levels for FXR and Shp were significantly decreased, and these changes could be prevented by co-treatment with the FXR agonist OCA. In silico molecular docking studies showed that INH, acetyl isoniazid, isonicotinic acid and PIH may be potential FXR inhibitors, and a TR-FRET FXR-coactivator assay confirmed that PIH is a strong antagonist of FXR (IC50 = 52 nM). To further determine if PIH also inhibits FXR activity in vivo, rats were treated with PIH directly (5 mg/kg). Liver total bile acids were significantly increased while FXR expression was not changed, but Shp mRNA levels were significantly decreased and Cyp7a1 mRNA was significantly increased, consistent with PIH acting as an FXR antagonist. In summary, PIH inhibition of liver FXR function leading to bile acid accumulation in hepatocytes may be an early pathogenesis event in INH-induced liver injury.

Disruption of hepatic small heterodimer partner induces dissociation of steatosis and inflammation in experimental nonalcoholic steatohepatitis

Disruption of hepatic small heterodimer partner induces dissociation of steatosis and inflammation in experimental nonalcoholic steatohepatitis

Hyperglycemia exacerbates dyslipidemia-induced changes in uptake, synthesis, and transporters of bile acids in rats: Assessment of restorative potentials of ALA and EPA + DHA

In this study, under dyslipidemia (D) and dyslipidemia with diabetes (DD) conditions, we assessed the intestinal bile acid (BA) uptake, BA transporter expression and de novo BA synthesis in rats. The restorative potentials n-3 fatty acids [{α-linolenic acid (ALA), DD+canola oil (CNO)} and eicosapentaenoic acid (EPA)+docosahexaenoic acid (DHA), DD+fish oil (FO)}] were also assessed. DD group had significantly (p < 0.05) increased intestinal BA uptake and Asbt and Osta/b mRNA expression, increased serum BA level, decreased hepatic Ntcp, Bsep and Cyp7a1 mRNA expression, increased hepatic HMG-CoA reductase activity, increased hepatic Fxr and Shp mRNA expression, decreased hepatic Lrh-1 and Hnf4a mRNA expression and decreased fecal BA level, when compared to control, D, DD+CNO, and DD+FO group. Our data show for the first time that, hyperglycemia exacerbates dyslipidemia-induced dysregulation of BA metabolism and transport. Dietary EPA+DHA exhibited superior effect than ALA in normalizing dyslipidemia and hyperglycemia-induced disturbances.

N-3 Fatty Acids Abrogate Dyslipidemia-Induced Changes in Bile Acid Uptake, Synthesis, and Transport in Young and Aged Dyslipidemic Rats.

In this study, the effect of n-3 fatty acids (FA) [α-linolenic acid (ALA) and eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA)] on the intestinal bile acid (BA) uptake, hepatic BA synthesis, and enterohepatic bile acid transporters (BAT) was assessed in young and aged dyslipidemic rats. Dyslipidemia was induced in young and aged rats by feeding a high-fat (HF) diet. Experimental groups received diets containing canola oil (HF + CNO) and fish oil (HF + FO) as a source of ALA and EPA + DHA, respectively. After 60 days of feeding, intestinal BA uptake and expression of apical sodium-dependent bile acid transporter (Asbt), organic solute transporter-alpha/beta (Osta/b) messenger RNA (mRNA), and hepatic expression of Na+ taurocholate cotransporting polypeptide (Ntcp), bile salt export pump (Bsep), cholesterol 7-α hydroxylase A1 (Cyp7a1), Farnesoid X receptor (Fxr), small heterodimer partner-1 (Shp), liver receptor homolog-1 (Lrh-1), and hepatic nuclear factor-4 alpha (Hnf4a) mRNA were measured. Hepatic 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase activity and total BA in serum, liver, and feces were assessed. The dyslipidemic HF group had: (1) increased intestinal BA uptake and Asbt and Osta/b mRNA expression, (2) increased BA in serum, (3) decreased hepatic expression of Ntcp, Bsep, and Cyp7a1 mRNA, (4) increased activity of HMG-CoA reductase, (5) increased hepatic expression of Fxr and Shp mRNA, (6) decreased hepatic expression of Lrh-1 and Hnf4a mRNA, and (7) decreased BA in feces, when compared to control, HF + CNO, and HF + FO groups. Immunostaining revealed increased expression of intestinal Asbt and hepatic Ntcp protein in the HF group when compared to control, HF + CNO, and HF + FO groups. n-3 FA abrogated dyslipidemia-induced changes in the intestinal uptake, hepatic synthesis, and enterohepatic transporters of BA in both young and aged rats. EPA + DHA was more effective than ALA in modulating dyslipidemia-induced changes.

Basal PPARα inhibits bile acid metabolism adaptation in chronic cholestatic model induced by α-naphthylisothiocyanate

Cholestasis is one of the most challenging diseases to be treated in current hepatology. However little is known about the adaptation difference and the underlying mechanism between acute and chronic cholestasis. In this study, wild-type and Pparα-null mice were orally administered diet containing 0.05% ANIT to induce chronic cholestasis. Biochemistry, histopathology and serum metabolome analysis exhibited the similar toxic phenotype between wild-type and Pparα-null mice. Bile acid metabolism was strongly adapted in Pparα-null mice but not in wild-type mice. The Shp and Fxr mRNA was found to be doubled in cholestatic Pparα-null mice compared with the control group. Western blot confirmed the up-regulated expression of FXR in Pparα-null mice treated with ANIT. Inflammation was found to be stronger in Pparα-null mice than those in wild-type mice in chronic cholestasis. These data chain indicated that bile acid metabolism and inflammation signaling were different between wild-type and Pparα-null mice developing chronic cholestasis, although their toxic phenotypes could not be discriminated. So basal PPARα cross-talked with FXR and inhibited bile acid metabolism adaptation in chronic cholestasis.

Aberrant expression of miR-141 and nuclear receptor small heterodimer partner in clinical samples of prostate cancer.

Prostate cancer (PCa) is the second most common cancer in men worldwide. Currently, prostate-specific antigen (PSA) test and digital rectal exam are the main screening tests used for PCa diagnosis. However, due to the low specificity of these tests, new alternative biomarkers such as deregulated RNAs and microRNAs have been implemented. Aberrant expressions of small heterodimer partner gene (SHP, NR0B2) and mir-141 are reported in various cancers. The aim of this study was to investigate the SHP and miR-141 expression level in tissue samples of prostate cancer. The expression level of SHP gene and miR-141 was assessed by real time PCR and their relative amounts were calculated by the Δ⁢ΔCT method. Also, IHC technique was used to determine the expression level of SHP protein. The miR-141 was significantly up-regulated in the samples of metastatic tumors compared to localized tumor samples (P< 0.001, 31.17-fold change). Tumor samples showed lower SHP mRNA expression levels than BPH samples (p= 0.014, 4.7-fold change). The results of paired t-test analysis showed there was no significant difference between the SHP gene expression in PCa samples and their matched tumor-adjacent normal tissue (p= 0.5). The data obtained in our study confirm the involvement of miR-141 in PCa progression and metastasis. These effects could be mediated by AR via down-regulation of its co-repressor protein, i.e., SHP.

Long noncoding RNA MEG3 induces cholestatic liver injury by interaction with PTBP1 to facilitate shp mRNA decay.

MEG3 causes cholestasis by serving as a guide RNA scaffold to recruit PTBP1 to destabilize Shp mRNA. SHP in turn represses CREB-mediated activation of MEG3 expression in a feedback-regulatory fashion. (Hepatology 2017;65:604-615).

Disrupted Murine Gut-to-Human Liver Signaling Alters Bile Acid Homeostasis in Humanized Mouse Liver Models.

The humanized liver mouse model is being exploited increasingly for human drug metabolism studies. However, its model stability, intercommunication between human hepatocytes and mouse nonparenchymal cells in liver and murine intestine, and changes in extrahepatic transporter and enzyme expressions have not been investigated. We examined these issues in FRGN [fumarylacetoacetate hydrolase (Fah-/-), recombination activating gene 2 (Rag2-/-), and interleukin 2 receptor subunit gamma (IL-2rg -/-) triple knockout] on nonobese diabetic (NOD) background] and chimeric mice: mFRGN and hFRGN (repopulated with mouse or human hepatocytes, respectively). hFRGN mice showed markedly higher levels of liver cholesterol, biliary bilirubin, and bile acids (liver, bile, and plasma; mainly human forms, but also murine bile acids) but lower transforming growth factor beta receptor 2 (TGFBR2) mRNA expression levels (10%) in human hepatocytes and other proliferative markers in mouse nonparenchymal cells (Tgf-β1) and cholangiocytes [plasma membrane-bound, G protein-coupled receptor for bile acids (Tgr5)], suggestive of irregular regeneration processes in hFRGN livers. Changes in gene expression in murine intestine, kidney, and brain of hFRGN mice, in particular, induction of intestinal farnesoid X receptor (Fxr) genes: fibroblast growth factor 15 (Fgf15), mouse ileal bile acid binding protein (Ibabp), small heterodimer partner (Shp), and the organic solute transporter alpha (Ostα), were observed. Proteomics revealed persistence of remnant murine proteins (cyotchrome P450 7α-hydroxylase (Cyp7a1) and other enzymes and transporters) in hFRGN livers and suggest the likelihood of mouse activity. When compared with normal human liver tissue, hFRGN livers showed lower SHP mRNA and higher CYP7A1 (300%) protein expression, consequences of tβ- and tα-muricholic acid-mediated inhibition of the FXR-SHP cascade and miscommunication between intestinal Fgf15 and human liver fibroblast growth factor receptor 4 (FGFR4), as confirmed by the unchanged hepatic pERK/total ERK ratio. Dysregulation of hepatocyte proliferation and bile acid homeostasis in hFRGN livers led to hepatotoxicity, gallbladder distension, liver deformity, and other extrahepatic changes, making questionable the use of the preparation for drug metabolism studies.

Highlights of This Issue

The rarity of RET oncofusions in lung cancer impairs the pace of clinical studies in this subtype. Huang and colleagues established a transgenic KIF5B–RET fusion lung cancer mouse model that can develop invasive lung adenocarcinoma with fibrotic desmoplasia. The authors also identified RETV804L gatekeeper and vandetanib-resistant RETG810A mutations in cultured cells with increasing concentrations of RET inhibitors. Ponatinib was identified as the most potent RET inhibitor, displayed hyperactivity to RETG810A, and caused lung tumor regression in the animals. These studies established preclinical models that can rapidly evaluate efficacy of RET inhibitors, discover resistance mechanisms, and identify approaches to thwart resistance.Therapeutically targeting intracellular kinases is biologically attractive but fundamentally challenging. Interpreting drug behavior in cellular and animal models is confounded by redundant enzymatic activities, common substrates, and cell-specific signaling networks. To better understand clinical characteristics, cyclin-dependent kinase drugs (abemaciclib, dinaciclib, palbociclib, ribociclib) were profiled using cellular (untransformed, tumorigenic), biophysical, and structural approaches. CDK–drug co-crystal structures coupled with biochemical analyses reveal a unique hinge architecture that enables kinome selectivity and the extent of CDK family engagement is derived from other ATP binding pocket interactions. Together, these findings provide insight into drug pharmacology and a framework for rational drug design.A liposome-based platform for the co-delivery of the 3rd generation P-gp inhibitor tariquidar and paclitaxel was developed to overcome Taxol resistance in ovarian cancer cells. Using several functional assays and imaging approaches, Zhang, Sriraman, and colleagues showed that the dual drug-loaded liposomes inhibited cell viability, blocked proliferation, and caused G2–M arrest in vitro. In a xenograft model of Taxol resistance ovarian cancer, the liposomes significantly impeded tumor growth through the induction of apoptosis and reduction of proliferation. This novel nanomedicine has potential for clinical testing in ovarian cancer patients with P-gp–overexpressing tumors.The expression of small heterodimer partner (SHP, NROB2) is downregulated in metastatic HCC and is negatively associated with patient survival in several types of aggressive and highly metastatic human cancers. In this study, Yang and colleagues performed small molecule microarrays and discovered a novel activator of SHP, namely DSHN. DSHN transcriptionally activated Shp mRNA, but also stabilized SHP protein by preventing its ubiquitination and degradation. In vivo and in vitro evidence indicated that DSHN inhibited HCC metastasis via SHP-mediated repression of Ccl2 signaling. This study highlights the potential use of SHP as a therapeutically tractable gene in preclinical and translational studies.

A Novel Small Molecule Activator of Nuclear Receptor SHP Inhibits HCC Cell Migration via Suppressing Ccl2.

Small heterodimer partner (SHP, NR0B2) is a nuclear orphan receptor without endogenous ligands. Due to its crucial inhibitory role in liver cancer, it is of importance to identify small molecule agonists of SHP. As such, we initiated a probe discovery effort to identify compounds capable of modulating SHP function. First, we performed binding assays using small molecule microarrays (SMM) and discovered 5-(diethylsulfamoyl)-3-hydroxynaphthalene-2-carboxylic acid (DSHN) as a novel activator of SHP. DSHN transcriptionally activated Shp mRNA, but also stabilized the SHP protein by preventing its ubiquitination and degradation. Second, we identified Ccl2 as a new SHP target gene by RNA-seq. We showed that activation of SHP by DSHN repressed Ccl2 expression and secretion by inhibiting p65 activation of CCL2 promoter activity, as demonstrated in vivo in Shp-/- mice and in vitro in HCC cells with SHP overexpression and knockdown. Third, we elucidated a strong inhibitory effect of SHP and DSHN on HCC cell migration and invasion by antagonizing the effect of CCL2. Lastly, by interrogating a publicly available database to retrieve SHP expression profiles from multiple types of human cancers, we established a negative association of SHP expression with human cancer metastasis and patient survival. In summary, the discovery of a novel small molecule activator of SHP provides a therapeutic perspective for future translational and preclinical studies to inhibit HCC metastasis by blocking Ccl2 signaling. Mol Cancer Ther; 15(10); 2294-301. ©2016 AACR.

Pharmacological Activation of Fxr Attenuates Pregnancy‐Induced Repression of Hepatic and Ileal Bile Acid Transporters in Mice

The Farnesoid X Receptor (Fxr) controls bile acid homeostasis by coordinately regulating the expression of bile acid synthesis enzymes (Cyp7a1) and transporters in the intestine (Asbt, Ostβ) and liver (Ntcp, Oatp1a4, Bsep, Mrp3, Ostβ), as well as endocrine (Fgf15) and transcription (Shp) factors. We have demonstrated that expression of bile acid synthesis enzymes and transporters is dysregulated in pregnant mice. To determine the role of Fxr in the adaptive gene responses of pregnancy, mRNA and protein expression were quantified in livers and intestines from wild‐type and Fxr‐null mice, as well as wild‐type mice administered the Fxr agonist GW4064 (GW). In response to pregnancy, ileal Fgf15, Asbt and Ostβ mRNAs decreased by 25 to 57% in wild‐type mice. Expression of these genes was also constitutively reduced 40 to 99% in Fxr‐null mice, although unchanged by pregnancy. In the liver, pregnancy‐mediated repression of Bsep mRNA was observed in wild‐type, but not Fxr‐null mice. Similar to wild‐types, pregnancy further reduced hepatic Shp, Ntcp, Oatp1a4 and Mrp3 mRNA levels of Fxr‐null mice. Treatment of pregnant mice with GW induced hepatic Shp, Ntcp, Bsep, Mrp3 and Ostβ, and ileal Fgf15 and Ostβ mRNA levels by 20 to 300%. GW also enhanced protein levels of Ntcp and Bsep in pregnant mice. Likewise, the induction of Cyp7a1 in pregnant mice was reduced by GW to levels lower than virgin controls. Collectively, these data implicate both intestinal and hepatic Fxr signaling in pregnancy‐related procholestatic gene changes, and suggest that Fxr activation may restore bile acid homeostasis in maternal cholestasis. Supported by AFPE, R01ES020522, GM104037 and T32ES007148.

Perturbations of fibroblast growth factors 19 and 21 in type 2 diabetes.

Fibroblast growth factors 19 and 21 (FGF19 and FGF21) have been implicated, independently, in type 2 diabetes (T2D) but it is not known if their circulating levels correlate with each other or whether the associated hepatic signaling mechanisms that play a role in glucose metabolism are dysregulated in diabetes. We used a cross-sectional, case/control, experimental design involving Class III obese patients undergoing Roux-en-Y bariatric surgery (RYGB), and measured FGF19 and FGF21 serum levels and hepatic gene expression (mRNA) in perioperative liver wedge biopsies. We found that T2D patients had lower FGF19 and higher FGF21 serum levels. The latter was corroborated transcriptionally, whereby, FGF21, as well as CYP7A1, β-Klotho, FGFR4, HNF4α, and glycogen synthase, but not of SHP or FXR mRNA levels in liver biopsies were higher in T2D patients that did not remit diabetes after RYGB surgery, compared to T2D patients that remitted diabetes after RYGB surgery or did not have diabetes. In a Phenome-wide association analysis using 205 clinical variables, higher FGF21 serum levels were associated with higher glucose levels and various cardiometabolic disease phenotypes. When serum levels of FGF19 were < 200 mg/mL and FGF21 > 500 mg/mL, 91% of patients had diabetes. These data suggest that FGF19/FGF21 circulating levels and hepatic gene expression of the associated signaling pathway are significantly dysregulated in type 2 diabetes.

Silencing the expression of the salivary sheath protein causes transgenerational feeding suppression in the aphid Sitobion avenae.

Aphids produce gel saliva during feeding which forms a sheath around the stylet as it penetrates through the apoplast. The sheath is required for the sustained ingestion of phloem sap from sieve elements and is thought to form when the structural sheath protein (SHP) is cross-linked by intermolecular disulphide bridges. We investigated the possibility of controlling aphid infestation by host-induced gene silencing (HIGS) targeting shp expression in the grain aphid Sitobion avenae. When aphids were fed on transgenic barley expressing shp double-stranded RNA (shp-dsRNA), they produced significantly lower levels of shp mRNA compared to aphids feeding on wild-type plants, suggesting that the transfer of inhibitory RNA from the plant to the insect was successful. shp expression remained low when aphids were transferred from transgenic plants and fed for 1 or 2weeks, respectively, on wild-type plants, confirming that silencing had a prolonged impact. Reduced shp expression correlated with a decline in growth, reproduction and survival rates. Remarkably, morphological and physiological aberrations such as winged adults and delayed maturation were maintained over seven aphid generations feeding on wild-type plants. Targeting shp expression therefore appears to cause strong transgenerational effects on feeding, development and survival in S.avenae, suggesting that the HIGS technology has a realistic potential for the control of aphid pests in agriculture.

Negative feedback loop of cholesterol regulation is impaired in the livers of patients with Alagille syndrome

AimTo characterize cholesterol regulation in the liver of patients with Alagille syndrome (AGS). MethodsSerum total cholesterol (TC) and total bile acid (TBA) levels were measured in 23 AGS patients. The expressions of genes involved in cholesterol regulation, including low-density lipoprotein receptor (LDLR), scavenger receptor class B type I (SR-BI), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), cholesterol 7α-hydroxylase (CYP7A1), ATP-binding cassette transporter (ABC) A1, and ABCG1/5/8, were measured in liver tissues from five of these patients. Expression of regulators for these genes, including farnesoid X receptor/small heterodimer partner (SHP), liver X receptor α (LXRα) and mature Sterol regulatory element-binding protein 2 (SREBP2) was measured. The expression of mature SREBP2 protein was also examined. ResultsSerum TC and TBA levels were correlated in the AGS patients. Liver cholesterol was also increased compared with controls, and correlated with bile acid contents. LDLR, SR-BI, HMGCR, and ABCGs mRNA expression were upregulated, while CYP7A1 mRNA expression was downregulated in AGS livers. SHP and LXRα mRNA expression was also increased, but maturation of SREBP2 was not suppressed in the patients. ConclusionsThe major upregulators of liver cholesterol might be increased in AGS patients, indicating an impaired negative feedback mechanism and accelerated liver cholesterol accumulation.