Amyloidogenic light chains impair plasma cell survival.

Systemic light chain amyloidosis (AL) is a clonal plasma cell disorder characterized by the deposition of misfolded immunoglobulin light chains (LC) as insoluble fibrils in organs. The lack of suitable models has hindered the investigation of the disease mechanisms. Our aim was to establish AL LC-producing plasma cell lines and use them to investigate the biology of the amyloidogenic clone. We used lentiviral vectors to generate cell lines expressing LC from patients suffering from AL amyloidosis. The AL LC-producing cell lines showed a significant decrease in proliferation, cell cycle arrest, and an increase in apoptosis and autophagy as compared with the multiple myeloma LC-producing cells. According to the results of RNA sequencing the AL LC-producing lines showed higher mitochondrial oxidative stress, and decreased activity of the Myc and cholesterol pathways. The neoplastic behavior of plasma cells is altered by the constitutive expression of amyloidogenic LC causing intracellular toxicity. This observation may explain the disparity in the malignant behavior of the amyloid clone compared to the myeloma clone. These findings should enable future in vitro studies and help delineate the unique cellular pathways of AL, thus expediting the development of specific treatments for patients with this disorder.

Amelioration of diabesity-induced colorectal ontogenesis by omega-3 fatty acids in mice.

Postnatal intestinal ontogenesis in an animal model of diabesity may recapitulate morphological and transduction features of diabesity-induced intestinal dysplasia and its amelioration by endogenous (n-3) polyunsaturated fatty acids (PUFA). Proliferation, differentiation, and transduction aspects of intestinal ontogenesis have been studied here in obese, insulin-resistant db/db mice, in fat-1 transgene coding for desaturation of (n-6) PUFA into (n-3) PUFA, in db/db crossed with fat-1 mice, and in control mice. Diabesity resulted in increased colonic proliferation and dedifferentiation of epithelial colonocytes and goblet cells, with increased colonic ß-catenin and hepatocyte nuclear factor (HNF)-4α transcriptional activities accompanied by enrichment in HNF-4α-bound (n-6) PUFA. In contrast, in fat-1 mice, colonic proliferation was restrained, accompanied by differentiation of crypt stem cells into epithelial colonocytes and goblet cells and by decrease in colonic ß-catenin and HNF-4α transcriptional activities, with concomitant enrichment in HNF-4α-bound (n-3) PUFA at the expense of (n-6) PUFA. Colonic proliferation and differentiation, the profile of ß-catenin and HNF-4α-responsive genes, and the composition of HNF-4α-bound PUFA of db/db mice reverted to wild-type by introducing the fat-1 gene into the db/db context. Suppression of intestinal HNF-4α activity by (n-3) PUFA may ameliorate diabesity-induced intestinal ontogenesis and offer an effective preventive modality for colorectal cancer.

Suppression of multiple myeloma by mitochondrial targeting.

Treatment of multiple myeloma (MM) aims at inducing cell apoptosis by surpassing the limited capacity of MM cells to cope with oxidative stress. MM cell survival may further be suppressed by limiting cellular cholesterol. Long-chain fatty acid analogs of the MEDICA series promote mitochondrial stress and inhibit cholesterol biosynthesis, thus prompting us to verify their efficacy and mode-of-action in suppressing MM cell survival, in comparison to bortezomib. MEDICA analog is shown here to effectively suppress survival of MM cells, and to inhibit growth of MM xenograft. Suppression of MM cell survival by MEDICA is accompanied by inhibition of the STAT3, MAPK and the mTORC1 transduction pathways due to mitochondrial oxidative stress. MEDICA-induced oxidative stress is abrogated by added exogenous cholesterol. Suppression of MM cell survival by bortezomib is similarly driven by bortezomib-induced oxidative stress, being abrogated by added cholesterol. In line with that, the time-to-best-response of MM patients to bortezomib-based treatment protocols is shown to be positively correlated with their plasma cholesterol level. MEDICA profile may indicate novel therapeutic potential in the management of MM.

Treatment of ErbB2 breast cancer by mitochondrial targeting.

BACKGROUND: ErbB2 breast cancer still remains an unmet need due to primary and/or acquired resistance to current treatment strategies. MEDICA compounds consist of synthetic long-chain α,ω-dicarboxylic acids previously reported to suppress breast cancer in PyMT transgenic mice. METHODS: MEDICA efficacy and mode of action in the ErbB2 context was studied in ErbB2 transgenic mice and human breast cancer cells. RESULTS: MEDICA treatment is shown here to suppress ErbB2 breast tumors and lung metastasis in ErbB2/neu MMTV transgenic mice, to suppress ErbB2/neu xenografts in nod/scid mice, and to suppress survival of AU565 and BT474 human ErbB2 breast cancer cells. Suppression of ErbB2 breast tumors by MEDICA is due to lipid raft disruption with loss of ErbB family members, including EGFR, ErbB2, and ErbB3. In addition, MEDICA inhibits mTORC1 activity, independently of abrogating the ErbB receptors and their signaling cascades. The double hit of MEDICA in abrogating ErbB and mTORC1 is partly accounted for by targeting mitochondria complex I. CONCLUSIONS: Mitochondrial targeting by MEDICA suppresses ErbB2 breast tumors and metastasis due to lipid raft disruption and inhibition of mTORC1 activity. Inhibition of mTORC1 activity by MEDICA avoids the resistance acquired by canonical mTORC1 inhibitors like rapalogs or mTOR kinase inhibitors.

Inhibition of colorectal cancer by targeting hepatocyte nuclear factor-4alpha.

Hepatocyte nuclear factor-4alpha (HNF-4alpha) serves as target for fatty acid nutrients and xenobiotic amphipathic carboxylates and may account for the differential effects of dietary fatty acids on colorectal cancer (CRC). The putative role played by HNF-4alpha in CRC has been verified here by evaluating the effect of HNF-4alpha antagonists and HNF-4alpha siRNA on CRC growth and proliferation in cultured CRC cells and xenotransplanted nude mice in vivo. HNF-4alpha ligand antagonists of the MEDICA series, namely, beta,beta'-tetramethylhexadecanedioic acid (M16betabeta) and gamma,gamma'-tetramethyloctadocanedioic acid (M18gammagamma) as well as HNF-4alpha siRNA are shown here to inhibit growth and proliferation of HT29 and Caco2 CRC cells, accompanied by increased subG1 cell population, downregulated PCNA, activation of caspase-3, upregulation of Bak and cytoplasmic cytochrome-c, and downregulation of Bcl-2 resulting in apoptotic death. Inhibition of CRC growth with concomitant apoptosis was further confirmed in nude mice xenotransplanted with HT29 CRC cells. CRC suppression by HNF-4alpha ligand antagonists and by HNF-4alpha siRNA was accounted for by suppression of HNF-4alpha transcription and protein expression. alpha,alpha'-tetrachlorotetradecanedioic acid (Cl-DICA), a MEDICA analogue that fails to suppress HNF-4alpha, was ineffective in suppressing growth of cultured or xenotransplanted HT29 CRC cells. Hence, increased transcriptional activity of HNF-4alpha converging onto genes coding for antiapoptotic oncogenes and cytokines may promote CRC development. Suppression of HNF-4alpha activity by natural or xenobiotic HNF-4alpha ligand antagonists or by HNF-4alpha siRNA may offer a treatment mode for CRC.

Negative autoregulation of HNF-4alpha gene expression by HNF-4alpha1.

HNF-4alpha (hepatocyte nuclear factor-4alpha) is required for tissue-specific expression of many of the hepatic, pancreatic, enteric and renal traits. Heterozygous HNF-4alpha mutants are inflicted by MODY-1 (maturity onset diabetes of the young type-1). HNF-4alpha expression is reported here to be negatively autoregulated by HNF-4alpha1 and to be activated by dominant-negative HNF-4alpha1. Deletion and chromatin immunoprecipitation analysis indicated that negative autoregulation by HNF-4alpha1 was mediated by its association with the TATA-less HNF-4alpha core promoter enriched in Sp1, but lacking DR-1 response elements. Also, negative autoregulation by HNF-4alpha1 was independent of its transactivation function, being similarly exerted by transcriptional-defective MODY-1 missense mutants of HNF-4alpha1, or under conditions of suppressing or enhancing HNF-4alpha activity by small heterodimer partner or by inhibiting histone deacetylase respectively. Negative autoregulation by HNF-4alpha1 was abrogated by overexpressed Sp1. Transcriptional suppression by HNF-4alpha1 independently of its transactivation function may extend the scope of its transcriptional activity to interference with docking of the pre-transcriptional initiation complex to TATA-less promoters.

Suppression of B-Raf(V600E) cancers by MAPK hyper-activation.

B-Raf(V600E) activates MEK/MAPK signalling and acts as oncogenic driver of a variety of cancers, including melanoma, colorectal and papillary thyroid carcinoma. Specific B-Raf(V600E) kinase inhibitors (e.g., Vemurafenib) prove initial efficacy in melanoma followed shortly by acquired resistance, while failing in most other B-Raf(V600E) cancers due to primary resistance. Resistance is due to acquired mutations in the Ras/Raf/MEK/MAPK pathway and/or other oncogenic drivers that bypass B-Raf(V600E). Surprisingly, hyper-activation of MAPK by inhibiting its protein phosphatase 2A by a synthetic long-chain fatty acid analogue (MEDICA), results in oncogene-induced growth arrest and apoptosis of B-Raf(V600E) cancer cells. Growth arrest is accompanied by MAPK-mediated serine/threonine phosphorylation and suppression of a variety of oncogenic drivers that resist treatment by B-Raf(V600E) kinase inhibitors, including ErbB members, c-Met, IGFR, IRS, STAT3 and Akt. The combined activities of mutated B-Raf and MEDICA are required for generating hyper-activated MAPK, growth arrest and apoptosis, implying strict specificity for mutated B-Raf cancer cells.

Transcriptional suppression of human microsomal triglyceride transfer protein by hypolipidemic insulin sensitizers.

Microsomal triglyceride transfer protein (MTP) catalyzes the assembly and secretion of liver triglyceride-rich lipoproteins. The human MTP (hMTP) promoter activity is reported here to be suppressed by HNF-4alpha ligand antagonists (e.g., Medica analogs) or by PPARgamma ligand agonists (e.g., thiazolidinediones), thus accounting for their hypolipidemic activity in humans. Suppression of liver hMTP by Medica analogs or by thiazolidinediones was mediated by the TAAA sequence that serves as non-canonical TATA box of the hMTP core promoter. MTP suppression was evident in the specific context of the wild type hMTP core promoter, but not in the context of the mutated rodent-conforming hMTP core promoter governed by a canonical TATA box conjoined with its proximal (-50/-38)DR-1 element. hMTP suppression by Medica analogs or thiazolidinediones mediated by hMTP TAAA was independent of HNF-4alpha or PPARgamma. hMTP suppression by Medica analogs, but not by thiazolidinediones, was further complemented by inhibition of HNF-4alpha transcriptional activity transduced by the distal (-83/-70)DR-1 element of hMTP promoter. hMTP promoter activity was unaffected by PPARalpha activation. Furthermore, in contrast to hMTP, the promoter activity of the rodent-conforming hMTP was robustly activated by Wy-14,643-activated PPARalpha or by thiazolidinedione-activated PPARgamma. Transcriptional activation by PPARalpha or PPARgamma of the rodent-conforming, but not the wild type hMTP gene promoter, resulted from the species-specific context of the respective proximal DR-1 elements. Hence, suppression of hMTP transcription by hypolipidemic insulin sensitizers requires the specific context of hMTP core promoter. In light of the species-specific context of MTP core promoters, the rodent MTP promoter may not substitute for the human promoter when searching for hypolipidemic MTP suppressors.

Stability of fatty acyl-coenzyme A thioester ligands of hepatocyte nuclear factor-4alpha and peroxisome proliferator-activated receptor-alpha.

Although long-chain fatty acyl-coenzyme A (LCFA-CoA) thioesters are specific high-affinity ligands for hepatocyte nuclear factor-4alpha (HNF-4alpha) and peroxisome proliferator-activated receptor-alpha (PPARalpha), X-ray crystals of the respective purified recombinant ligand-binding domains (LBD) do not contain LCFA-CoA, but instead exhibit bound LCFA or have lost all ligands during the purification process, respectively. As shown herein: (i) The acyl chain composition of LCFA bound to recombinant HNF-4alpha reflected that of the bacterial LCFA-CoA pool, rather than the bacterial LCFA pool. (ii) Bacteria used to produce the respective HNF-4alpha and PPARalpha contained nearly 100-fold less LCFA-CoA than LCFA. (iii) Under conditions used to crystallize LBD (at least 3 wk at room temperature in aqueous buffer), 16:1-CoA was very unstable in buffer alone. (iv) In the presence of the respective nuclear receptor (i.e., HNF-4alpha and PPARalpha), LBD 70-75% of 16:1-CoA was degraded after 1 d at room temperature in the crystallization buffer, whereas as much as 94-97% of 16:1-CoA was degraded by 3 wk. (v) Cytoplasmic LCFA-CoA binding proteins such as acyl-CoA binding protein, sterol carrier protein-2, and liver-FA binding protein slowed the process of 16:1-CoA degradation proportional to their respective affinities for this ligand. Taken together, these data for the first time indicated that the absence of LCFA-CoA in the crystallized HNF-4alpha and PPARalpha was due to the paucity of LCFA-CoA in bacteria as well as to the instability of LCFA-CoA in aqueous buffers and the conditions used for LBD crystallization. Furthermore, instead of protecting bound LCFA-CoA from autohydrolysis like several cytoplasmic LCFA-CoA binding proteins, these nuclear receptors facilitated LCFA-CoA degradation.

Kinase-independent transcriptional co-activation of peroxisome proliferator-activated receptor alpha by AMP-activated protein kinase.

AMPK (AMP-activated protein kinase) responds to intracellular ATP depletion, while PPARalpha (peroxisome proliferator-activated receptor alpha) induces the expression of genes coding for enzymes and proteins involved in increasing cellular ATP yields. PPARalpha-mediated transcription is shown here to be co-activated by the alpha subunit of AMPK, as well as by kinase-deficient (Thr172Ala) and kinase-less (Asp157Ala, Asp139Ala) mutants of AMPKalpha. The Ser452Ala mutant of mPPARalpha mutated in its putative consensus AMPKalpha phosphorylation site is similarly co-activated by AMPKalpha. AMPKalpha or its kinase-less mutants bind to PPARalpha; binding is increased by MgATP, to a lesser extent by MgADP, but not at all by AMP or ZMP [AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) monophosphate]. ATP-activated binding of AMPKalpha to PPARalpha is mediated primarily by the C-terminal regulatory domain of AMPKalpha. PPARalpha co-activation by AMPKalpha may, however, require its secondary interaction with the N-terminal catalytic domain of AMPKalpha, independently of its kinase activity. While AMPK catalytic activity is activated by AICAR, PPARalpha co-activation and PPARalpha-controlled transcription are robustly inhibited by AICAR, with concomitant translocation of nuclear AMPKalpha or its kinase-less mutants to the cytosol. In conclusion, AMPKalpha, independently of its kinase activity, co-activates PPARalpha both in primary rat hepatocytes and in PPARalpha-transfected cells. The kinase and transcriptional co-activation modes of AMPKalpha are both regulated by the cellular ATP/AMP ratio. Co-activation of PPARalpha by AMPKalpha may transcriptionally complement AMPK in maintaining cellular ATP status.

Long-chain fatty acid analogues suppress breast tumorigenesis and progression.

Obesity and type 2 diabetes (T2D) are associated with increased breast cancer incidence and mortality, whereas carbohydrate-restricted ketogenic diets ameliorate T2D and suppress breast cancer. These observations suggest an inherent efficacy of nonesterified long-chain fatty acids (LCFA) in suppressing T2D and breast tumorigenesis. In this study, we investigated novel antidiabetic MEDICA analogues consisting of methyl-substituted LCFA that are neither ß-oxidized nor esterified to generate lipids, prompting interest in their potential efficacy as antitumor agents in the context of breast cancer. In the MMTV-PyMT oncomouse model of breast cancer, in which we confirmed that tumor growth could be suppressed by a carbohydrate-restricted ketogenic diet, MEDICA treatment suppressed tumor growth, and lung metastasis, promoting a differentiated phenotype while suppressing mesenchymal markers. In human breast cancer cells, MEDICA treatment attenuated signaling through the STAT3 and c-Src transduction pathways. Mechanistic investigations suggested that MEDICA suppressed c-Src-transforming activity by elevating reactive oxygen species production, resulting in c-Src oxidation and oligomerization. Our findings suggest that MEDICA analogues may offer therapeutic potential in breast cancer and overcome the poor compliance of patients to dietary carbohydrate restriction.

Prevention of diabetes-promoted colorectal cancer by (n-3) polyunsaturated fatty acids and (n-3) PUFA mimetic.

The global obesity / diabetes epidemic has resulted in robust increase in the incidence of colorectal cancer (CRC). Epidemiological, animal and human studies have indicated efficacy of (n-3) PUFA in chemoprevention of sporadic and genetic-driven CRC. However, diabetes-promoted CRC presents a treatment challenge that surpasses that of sporadic CRC. This report analyzes the efficacy of (n-3) PUFA generated by the fat-1 transgene that encodes an (n-6) to (n-3) PUFA desaturase, and of synthetic (n-3) PUFA mimetic (MEDICA analog), to suppress CRC development in carcinogen-induced diabetes-promoted animal model. Carcinogen-induced CRC is shown here to be promoted by the diabetes context, in terms of increased aberrant crypt foci (ACF) load, cell proliferation and epithelial dedifferentiation, being accompanied by increase in the expression of HNF4α, ß-catenin, and ß-catenin-responsive genes. Incorporating the fat-1 transgene in the diabetes context, or oral MEDICA treatment, resulted in ameliorating the diabetic phenotype and in abrogating CRC, with decrease in ACF load, cell proliferation and the expression of HNF-4α, ß-catenin, and ß-catenin-responsive genes. The specificity of (n-3) PUFA in abrogating CRC development, as contrasted with enhancing CRC by (n-6) PUFA, was similarly verified in CRC cell lines. These findings may indicate prospective therapeutic potential of (n-3) PUFA or MEDICA in the management of CRC, in particular diabetes-promoted CRC.

PF-4708671 activates AMPK independently of p70S6K1 inhibition.

The P70 ribosomal protein S6 kinase 1 (P70S6K1) is activated by the mammalian target of rapamycin (mTORC1) and regulates proliferation, growth, and metabolism. PF-4708671 is a novel, cell-permeable, has been proposed to be a highly specific inhibitor of p70S6K1. It is used in micromolar concentration range to dissect signaling pathways downstream of mTORC1 and to study the function of p70S6K1. Here we show that PF-4708671 induces AMP-activated protein kinase (AMPK) phosphorylation and activation in immortalized mouse embryonic fibroblasts (MEF) independently of p70S6K1, due to specific inhibition of mitochondrial respiratory chain Complex I.

Suppression of FoxO1 activity by long-chain fatty acyl analogs.

OBJECTIVE: Overactivity of the Forkhead transcription factor FoxO1 promotes diabetic hyperglycemia, dyslipidemia, and acute-phase response, whereas suppression of FoxO1 activity by insulin may alleviate diabetes. The reported efficacy of long-chain fatty acyl (LCFA) analogs of the MEDICA series in activating AMP-activated protein kinase (AMPK) and in treating animal models of diabesity may indicate suppression of FoxO1 activity. RESEARCH DESIGN AND METHODS: The insulin-sensitizing and anti-inflammatory efficacy of a MEDICA analog has been verified in guinea pig and in human C-reactive protein (hCRP) transgenic mice, respectively. Suppression of FoxO1 transcriptional activity has been verified in the context of FoxO1- and STAT3-responsive genes and compared with suppression of FoxO1 activity by insulin and metformin. RESULTS: Treatment with MEDICA analog resulted in total body sensitization to insulin, suppression of lipopolysaccharide-induced hCRP and interleukin-6-induced acute phase reactants and robust decrease in FoxO1 transcriptional activity and in coactivation of STAT3. Suppression of FoxO1 activity was accounted for by its nuclear export by MEDICA-activated AMPK, complemented by inhibition of nuclear FoxO1 transcriptional activity by MEDICA-induced C/EBPß isoforms. Similarly, insulin treatment resulted in nuclear exclusion of FoxO1 and further suppression of its nuclear activity by insulin-induced C/EBPß isoforms. In contrast, FoxO1 suppression by metformin was essentially accounted for by its nuclear export by metformin-activated AMPK. CONCLUSIONS: Suppression of FoxO1 activity by MEDICA analogs may partly account for their antidiabetic anti-inflammatory efficacy. FoxO1 suppression by LCFA analogs may provide a molecular rational for the beneficial efficacy of carbohydrate-restricted ketogenic diets in treating diabetes.

AMPK activation by long chain fatty acyl analogs.

The antidiabetic efficacy of first-line insulin sensitizers (e.g., metformin, glitazones) is accounted for by activation of AMP-activated protein kinase (AMPK). Long chain fatty acids (LCFA) activate AMPK, but their putative antidiabetic efficacy is masked by their beta-oxidized or esterified lipid products. Substituted alpha,omega-dicarboxylic acids of 14-18 carbon atoms in length (MEDICA analogs) are not metabolized beyond their acyl-CoA thioesters, and may therefore simulate AMPK activation by LCFA while avoiding LCFA turnover into beta-oxidized or esterified lipid products. MEDICA analogs are shown here to activate AMPK and some of its downstream targets in vivo, in cultured cells and in a cell-free system consisting of the (alpha(1)beta(1)gamma(1))AMPK recombinant and LKB1-MO25-STRAD (AMPK-kinase) recombinant proteins. AMPK activation by MEDICA is accompanied by normalizing the hyperglycemia-hyperinsulinemia of diabetic db/db mice in vivo with suppression of hepatic glucose production in cultured liver cells. Activation of AMPK by MEDICA or LCFA is accounted for by (a) decreased intracellular ATP/AMP ratio and energy charge by the free acid, (b) activation of LKB1 phosphorylation of AMPK(Thr172) by the acyl-CoA thioester. The two activation modes are complementary since LKB1/AMPK activation by the CoA-thioester is fully evident under conditions of excess AMP. MEDICA analogs may expand the arsenal of AMPK activators used for treating diabetes type 2.

Role of regulatory F-domain in hepatocyte nuclear factor-4alpha ligand specificity.

The F-domain of rat HNF-4alpha1 has a crucial impact on the ligand binding affinity, ligand specificity and secondary structure of HNF-4alpha. (i) Fluorescent binding assays indicate that wild-type, full-length HNF-4alpha (amino acids 1-455) has high affinity (Kd=0.06-12 nm) for long chain fatty acyl-CoAs (LCFA-CoA) and low affinity (Kd=58-296 nm) for unesterified long chain fatty acids (LCFAs). LCFA-CoA binding was due to close molecular interaction as shown by fluorescence resonance energy transfer (FRET) from full-length HNF-4alpha tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor), which yielded an intermolecular distance of 33 A, although no FRET to cis-parinaric acid was detected. (ii) Deleting the N-terminal A-D-domains, comprising the AF1 and DNA binding functions, only slightly affected affinities for LCFA-CoAs (Kd=0.9-4 nm) and LCFAs (Kd=93-581 nm). (iii) Further deletion of the F-domain robustly reduced affinities for LCFA-CoA and reversed ligand specificity (i.e. high affinity for LCFAs (Kd=1.5-32 nm) and low affinity for LCFA-CoAs (Kd=54-302 nm)). No FRET from HNF-4alpha-E (amino acids 132-370) tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor) was detected, whereas an intermolecular distance of 28 A was calculated from FRET between HNF-4alpha-E and cis-parinaric acid. (iv) Circular dichroism showed that LCFA-CoA, but not LCFA, altered the secondary structure of HNF-4alpha only when the F-domain was present. (v) cis-Parinaric acid bound to HNF-4alpha with intact F-domain was readily displaceable by S-hexadecyl-CoA, a nonhydrolyzable thioether analogue of LCFA-CoAs. Truncation of the F-domain significantly decreased cis-parinaric acid displacement. Hence, the C-terminal F-domain of HNF-4alpha regulated ligand affinity, ligand specificity, and ligand-induced conformational change of HNF-4alpha. Thus, characteristics of F-domain-truncated mutants may not reflect the properties of full-length HNF-4alpha.

Transcriptional regulation of human microsomal triglyceride transfer protein by hepatocyte nuclear factor-4alpha.

Microsomal triglyceride transfer protein (MTP) catalyzes the assembly of triglyceride (TG)-rich apolipoprotein B-containing liver (e.g., VLDL) and intestinal (e.g., chylomicron) lipoproteins. The human MTP gene promoter is reported here to associate in vivo with endogenous hepatocyte nuclear factor-4alpha (HNF-4alpha) and to be transactivated or transsuppressed by overexpressed or by dominant negative HNF-4alpha, respectively. Human MTP (hMTP) transactivation by HNF-4alpha is accounted for by the concerted activity of distal (-83/-70) and proximal (-50/-38) direct repeat 1 elements of the hMTP promoter that bind HNF-4alpha. Transactivation by HNF-4alpha is specifically antagonized by chicken ovalbumin upstream promoter. Transcriptional activation of hMTP by HNF-4alpha is mediated by HNF-4alpha domains engaged in ligand binding and ligand-driven transactivation and is further complemented by HNF-4alpha/HNF-1alpha synergism that involves the HNF-4alpha activation function 1 (AF-1) domain. hMTP transactivation by HNF-4alpha is specifically inhibited by beta,beta-tetramethyl-hexadecanedioic acid acting as an HNF-4alpha antagonist ligand. hMTP transactivation by HNF-4alpha may account for the activation or inhibition of MTP expression and the production of TG-rich lipoproteins by agonist (e.g., saturated fatty acids) or antagonist [e.g., (n-3) PUFA, hypolipidemic fibrates, or Methyl-substituted dicarboxylic acid (Medica) compounds] HNF-4alpha ligands.

Thioesterase activity and acyl-CoA/fatty acid cross-talk of hepatocyte nuclear factor-4{alpha}.

Hepatocyte nuclear factor-4alpha (HNF-4alpha) activity is modulated by natural and xenobiotic fatty acid and fatty acyl-CoA ligands as a function of their chain length, unsaturation, and substitutions. The acyl-CoA site of HNF-4alpha is reported here to consist of the E-F domain, to bind long-chain acyl-CoAs but not the respective free acids, and to catalyze the hydrolysis of bound fatty acyl-CoAs. The free acid pocket, previously reported in the x-ray structure of HNF-4alpha E-domain, entraps fatty acids but excludes acyl-CoAs. The acyl-CoA and free acid sites are distinctive and noncongruent. Free fatty acid products of HNF-4alpha thioesterase may exchange with free acids entrapped in the fatty acid pocket of HNF-4alpha. Cross-talk between the acyl-CoA and free fatty acid binding sites is abrogated by high affinity, nonhydrolyzable acyl-CoA ligands of HNF-4alpha that inhibit its thioesterase activity. Hence, HNF-4alpha transcriptional activity is controlled by its two interrelated acyl ligands and two binding sites interphased in tandem by the thioesterase activity. The acyl-CoA/free-acid and receptor/enzyme duality of HNF-4alpha extends the paradigm of nuclear receptors.

Ligand specificity and conformational dependence of the hepatic nuclear factor-4alpha (HNF-4alpha ).

Hepatic nuclear factor-4alpha (HNF-4alpha) controls the expression of genes encoding proteins involved in lipid and carbohydrate metabolism. Fatty acyl-CoA thioesters have recently been proposed to be naturally occurring ligands of HNF-4alpha and to regulate its transcriptional activity as function of their chain length and degree of unsaturation (Hertz, R., Magenheim, J., Berman, I., and Bar-Tana, J. (1998) Nature 392, 512-516). However, the apparent low affinities (microm K(d) values) obtained with a radiolabeled fatty acyl-CoA ligand binding assay raised questions regarding the physiological significance of this finding. Furthermore, it is not known whether interaction with fatty acyl-CoA alters the structure of HNF-4alpha. These issues were examined using rat recombinant HNF-4alpha ligand-binding domain (HNF-4alphaLBD) in conjunction with photon counting fluorescence and circular dichroism. First, fluorescence resonance energy transfer between HNF-4alphaLBD tryptophan (Trp) and cis-parinaroyl-CoA yielded an intermolecular distance of

Activation of the insulin gene promoter through a direct effect of hepatocyte nuclear factor 4 alpha.

Maturity onset diabetes of the young, subtype 1 (MODY1), is associated with defective glucose-dependent insulin secretion from pancreatic beta cells. MODY1 is caused by mutation in the transcription factor hepatocyte nuclear factor 4 alpha (HNF4 alpha). To understand better the MODY1 phenotype, we tested whether HNF4 alpha was able to modulate directly the insulin gene promoter. Transfection of cultured 293T cells with an HNF4 alpha expression vector led to 10-fold activation of a cotransfected reporter plasmid containing the rat insulin I gene promoter. Computer analysis revealed a potential HNF4 alpha-binding site between nucleotides -57 and -69 of the promoter; mutation of this sequence led to reduced ability of HNF4 alpha to activate the promoter. The ability of HNF4 alpha to bind this sequence was confirmed using gel shift analysis. In transfected INS-1 beta cells, mutation of either the HNF1 alpha site or the HNF4 alpha site in the insulin gene promoter led to 50-75% reduction in reporter gene activity; expression of dominant negative HNF4 alpha led to significant reduction in the activity of wild type and both mutated promoters. Thus, in addition to the previously described indirect action of HNF4 alpha on insulin gene expression mediated through elevated HNF1 alpha levels, HNF4 alpha also activates the insulin gene directly, through a previously unrecognized cis element.