Methylotroph bacteria and cellular metabolite carotenoid alleviate ultraviolet radiation-driven abiotic stress in plants.

Increasing UV radiation in the atmosphere due to the depletion of ozone layer is emerging abiotic stress for agriculture. Although plants have evolved to adapt to UV radiation through different mechanisms, but the role of phyllosphere microorganisms in counteracting UV radiation is not well studied. The current experiment was undertaken to evaluate the role of phyllosphere Methylobacteria and its metabolite in the alleviation of abiotic stress rendered by ultraviolet (UV) radiation. A potential pink pigmenting methylotroph bacterium was isolated from the phylloplane of the rice plant (oryzae sativa). The 16S rRNA gene sequence of the bacterium was homologous to the Methylobacter sp. The isolate referred to as Methylobacter sp N39, produced beta-carotene at a rate (µg ml-1 d-1) of 0.45-3.09. Biosynthesis of beta-carotene was stimulated by brief exposure to UV for 10 min per 2 days. Carotenoid biosynthesis was predicted as y = 3.09 × incubation period + 22.151 (r 2 = 0.90). The carotenoid extract of N39 protected E. coli from UV radiation by declining its death rate from 14.67% min-1 to 4.30% min-1 under UV radiation. Application of N39 cells and carotenoid extract also protected rhizobium (Bradyrhizobium japonicum) cells from UV radiation. Scanning electron microscopy indicated that the carotenoid extracts protected E. coli cells from UV radiation. Foliar application of either N39 cells or carotenoid extract enhanced the plant's (Pigeon pea) resistance to UV irradiation. This study highlight that Methylobacter sp N39 and its carotenoid extract can be explored to manage UV radiation stress in agriculture.

Identification of a novel function of hepatic long-chain acyl-CoA synthetase-1 (ACSL1) in bile acid synthesis and its regulation by bile acid-activated farnesoid X receptor.

Long-chain acyl-CoA synthetase 1 (ACSL1) plays a pivotal role in fatty acid ß­oxidation in heart, adipose tissue and skeletal muscle. However, key functions of ACSL1 in the liver remain largely unknown. We investigated acute effects of hepatic ACSL1 deficiency on lipid metabolism in adult mice under hyperlipidemic and normolipidemic conditions. We knocked down hepatic ACSL1 expression using adenovirus expressing a ACSL1 shRNA (Ad-shAcsl1) in mice fed a high-fat diet or a normal chow diet. Hepatic ACSL1 depletion generated a hypercholesterolemic phenotype in mice fed both diets with marked elevations of total cholesterol, LDL-cholesterol and free cholesterol in circulation and accumulations of cholesterol in the liver. Furthermore, SREBP2 pathway in ACSL1 depleted livers was severely repressed with a 50% reduction of LDL receptor protein levels. In contrast to the dysregulated cholesterol metabolism, serum triglycerides, free fatty acid and phospholipid levels were unaffected. Mechanistic investigations of genome-wide gene expression profiling and pathway analysis revealed that ACSL1 depletion repressed expressions of several key enzymes for bile acid biosynthesis, consequently leading to reduced liver bile acid levels and altered bile acid compositions. These results are the first demonstration of a requisite role of ACSL1 in bile acid biosynthetic pathway in liver tissue. Furthermore, we discovered that Acsl1 is a novel molecular target of the bile acid-activated farnesoid X receptor (FXR). Activation of FXR by agonist obeticholic acid repressed the expression of ACSL1 protein and mRNA in the liver of FXR wild-type mice but not in FXR knockout mice.

Farnesoid X Receptor Activation by Obeticholic Acid Elevates Liver Low-Density Lipoprotein Receptor Expression by mRNA Stabilization and Reduces Plasma Low-Density Lipoprotein Cholesterol in Mice.

Objective- The objective of this study was to determine whether and how activation of farnesoid X receptor (FXR) by obeticholic acid (OCA), a clinical FXR agonist, modulates liver low-density lipoprotein receptor (LDLR) expression under normolipidemic conditions. Approach and Results- Administration of OCA to chow-fed mice increased mRNA and protein levels of LDLR in the liver without affecting the sterol-regulatory element binding protein pathway. Profiling of known LDLR mRNA-binding proteins demonstrated that OCA treatment did not affect expressions of mRNA degradation factors hnRNPD (heterogeneous nuclear ribonucleoprotein D) or ZFP36L1 but increased the expression of Hu antigen R (HuR) an mRNA-stabilizing factor. Furthermore, inducing effects of OCA on LDLR and HuR expression were ablated in Fxr-/- mice. To confirm the post-transcriptional mechanism, we used transgenic mice (albumin-luciferase-untranslated region) that express a human LDLR mRNA 3' untranslated region luciferase reporter gene in the liver. OCA treatment led to significant rises in hepatic bioluminescence signals, Luc-untranslated region chimeric mRNA levels, and endogenous LDLR protein abundance, which were accompanied by elevations of hepatic HuR mRNA and protein levels in OCA-treated transgenic mice. In vitro studies conducted in human primary hepatocytes and HepG2 cells demonstrated that FXR activation by OCA and other agonists elicited the same inducing effect on LDLR expression as in the liver of normolipidemic mice. Furthermore, depletion of HuR in HepG2 cells by short interfering RNA transfection abolished the inducing effect of OCA on LDLR expression. Conclusions- Our study is the first to demonstrate that FXR activation increases LDLR expression in liver tissue by a post-transcriptional regulatory mechanism involving LDLR mRNA-stabilizing factor HuR.

Hepatic HNF1 transcription factors control the induction of PCSK9 mediated by rosuvastatin in normolipidemic hamsters.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) impedes low­density lipoprotein (LDL) receptor (LDLR)-mediated LDL-cholesterol uptake and has hence emerged as a critical regulator of serum cholesterol levels and a new therapeutic target for the treatment of hypercholesterolemia. Statins have been shown to elevate circulating PCSK9 levels by stimulating PCSK9 gene transcription, which reduces the clinical efficacy of statin in LDL­cholesterol reduction. The transcription of PCSK9 is partially controlled by the hepatocyte nuclear factor 1 (HNF1) binding site embedded in the proximal region of its promoter. In this study, we utilized adenoviral shRNA delivery vectors to generate liver-specific knockdown of HNF1α (Ad­shHNF1α) or HNF1ß (Ad­shHNF1ß) in hamsters to examine the impact of reduced hepatic expression of HNF1 transcription factors on statin­induced elevation of PCSK9 expression and serum cholesterol levels. We showed that the administration of rosuvastatin (RSV) to normolipidemic hamsters significantly augmented hepatic PCSK9 expression and serum PCSK9 levels. In addition, RSV treatment increased hepatic HNF1α protein levels without a clear effect on HNF1α mRNA expression. Injection of Ad-shHNF1α or Ad­shHNF1ß into hamsters both blunted RSV­induced elevation of PCSK9 serum concentration and hepatic mRNA and protein levels, which led to significant increases in liver LDLR protein abundance. Furthermore, hepatic depletion of HNF1 factors lowered circulating total cholesterol and non­high density lipoprotein cholesterol levels in RSV­treated hamsters. Our study demonstrates that both HNF1α and HNF1ß are positive regulators of hepatic PCSK9 transcription in hamster species and that transient, liver-specific knockdown of either HNF1α or HNF1ß could antagonize the RSV­induced elevation of serum PCSK9 and reduce circulating cholesterol levels.

Global warming potential and greenhouse gas emission under different soil nutrient management practices in soybean-wheat system of central India.

Soil nutrient management is a key component contributing to the greenhouse gas (GHG) flux and mitigation potential of agricultural production systems. However, the effect of soil nutrient management practices on GHG flux and global warming potential (GWP) is less understood in agricultural soils of India. The present study was conducted to compare three nutrient management systems practiced for nine consecutive years in a soybean-wheat cropping system in the Vertisols of India, in terms of GHG flux and GWP. The treatments were composed of 100% organic (ONM), 100% inorganic (NPK), and integrated nutrient management (INM) with 50% organic + 50% inorganic inputs. The gas samples for GHGs (CO2, CH4, and N2O) were collected by static chamber method at about 15-day interval during 2012-13 growing season. The change in soil organic carbon (SOC) content was estimated in terms of the changes in SOC stock in the 0-15 cm soil over the 9-year period covering 2004 to 2013. There was a net uptake of CH4 in all the treatments in both soybean and wheat crop seasons. The cumulative N2O and CO2 emissions were in the order of INM > ONM > NPK with significant difference between treatments (p < 0.05) in both the crop seasons. The annual GWP, expressed in terms of CH4 and N2O emission, also followed the same trend and was estimated to be 1126, 1002, and 896 kg CO2 eq ha-1 year-1 under INM, ONM, and NPK treatments, respectively. However, the change in SOC stock was significantly higher under ONM (1250 kg ha-1 year-1) followed by INM (417 kg ha-1 year-1) and least under NPK (198 kg ha-1 year-1) treatment. The wheat equivalent yield was similar under ONM and INM treatments and was significantly lower under NPK treatment. Thus, the GWP per unit grain yield was lower under ONM followed by NPK and INM treatments and varied from 250, 261, and 307 kg CO2 eq Mg-1 grain yield under ONM, NPK, and INM treatments, respectively.

Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ.

Peroxisome proliferator-activated receptor δ (PPARδ) regulates many genes involved in lipid metabolism. Hepatic lysophosphatidylcholine acyltransferase 3 (LPCAT3) has critical functions in triglycerides transport and endoplasmic reticulum stress response due to its unique ability to catalyze the incorporation of polyunsaturated fatty acids into phospholipids. Previous studies identified liver X receptor as the transcription factor controlling LPCAT3 expression in mouse liver tissue. Here we show that the hepatic LPCAT3 gene is transcriptionally regulated by PPARδ. Adenovirus-mediated knockdown of PPARδ in cultured hepatic cells and liver tissue reduced LPCAT3 mRNA levels, and exogenous overexpression of PPARδ increased LPCAT3 mRNA expression. Activation of PPARδ in HepG2, Huh7, and Hepa 1-6 cells with its specific agonists increased LPCAT3 mRNA levels in all three hepatic cell lines. Through conducting sequence analysis, LPCAT3 promoter assays, and direct DNA binding assays, we have mapped the functional PPAR-responsive element to a proximal region from -135 to -123 of the LPCAT3 promoter that plays an essential role in mediating PPARδ-induced transactivation of the LPCAT3 gene. Finally, we have provided in vivo evidence showing that activation of PPARδ by agonist L165041 in mice increased hepatic LPCAT3 mRNA abundance and LPCAT enzymatic activity, which is associated with increased incorporations of arachidonate into liver phosphatidylcholine and phosphatidylethanolamine. Furthermore, transient liver-specific knockdown of LPCAT3 in mice affected PPARδ-mediated activation of several hepatic genes involving in FA metabolism. Altogether, our new findings identify LPCAT3 as a direct PPARδ target gene and suggest a novel function of PPARδ in regulation of phospholipid metabolism through LPCAT3.

SREBP2 Activation Induces Hepatic Long-chain Acyl-CoA Synthetase 1 (ACSL1) Expression in Vivo and in Vitro through a Sterol Regulatory Element (SRE) Motif of the ACSL1 C-promoter.

Long-chain acyl-CoA synthetase 1 (ACSL1) plays a key role in fatty acid metabolism. To identify novel transcriptional modulators of ACSL1, we examined ACSL1 expression in liver tissues of hamsters fed a normal diet, a high fat diet, or a high cholesterol and high fat diet (HCHFD). Feeding hamsters HCHFD markedly reduced hepatic Acsl1 mRNA and protein levels as well as acyl-CoA synthetase activity. Decreases in Acsl1 expression strongly correlated with reductions in hepatic Srebp2 mRNA level and mature Srebp2 protein abundance. Conversely, administration of rosuvastatin (RSV) to hamsters increased hepatic Acsl1 expression. These new findings were reproduced in mice treated with RSV or fed the HCHFD. Furthermore, the RSV induction of acyl-CoA activity in mouse liver resulted in increases in plasma and hepatic cholesterol ester concentrations and reductions in free cholesterol amounts. Investigations on different ACSL1 transcript variants in HepG2 cells revealed that the mRNA expression of C-ACSL1 was specifically regulated by the sterol regulatory element (SRE)-binding protein (SREBP) pathway, and RSV treatment increased the C-ACSL1 abundance from a minor mRNA species to an abundant transcript. We analyzed 5'-flanking sequence of exon 1C of the human ACSL1 gene and identified one putative SRE site. By performing a promoter activity assay and DNA binding assays, we firmly demonstrated the key role of this SRE motif in SREBP2-mediated activation of C-ACSL1 gene transcription. Finally, we demonstrated that knockdown of endogenous SREBP2 in HepG2 cells lowered ACSL1 mRNA and protein levels. Altogether, this work discovered an unprecedented link between ACSL1 and SREBP2 via the specific regulation of the C-ACSL1 transcript.

A novel peroxisome proliferator response element modulates hepatic low-density lipoprotein receptor gene transcription in response to PPARδ activation.

The hepatic expression of low-density lipoprotein (LDL) receptor (LDLR) gene is regulated primarily at the transcriptional level by a sterol-regulatory element (SRE) in its proximal promoter region which is the site of action of SRE-binding protein 2 (SREBP2). However whether additional cis-regulatory elements contribute to LDLR transcription has not been fully explored. We investigated the function of a putative peroxisome proliferator-activated receptor (PPAR)-response element (PPRE) sequence motif located at -768 to -752 bases upstream of the transcription start site of human LDLR gene in response to PPARδ activation. Promoter luciferase reporter analyses showed that treating HepG2 cells with PPARδ agonist L165041 markedly increased the activity of a full-length LDLR promoter construct (pLDLR-1192) without any effects on the shorter promoter reporter pLDLR-234 that contains only the core regulatory elements SRE-1 and SP1 sites. Importantly, mutation of the PPRE sequence greatly attenuated the induction of the full-length LDLR promoter activity by L165041 without affecting rosuvastatin (RSV)-mediated transactivation. EMSA and ChIP assay further confirmed the binding of PPARδ to the LDLR-PPRE site. Treating HepG2 cells with L165041 elevated the mRNA and protein expressions of LDLR without affecting the LDLR mRNA decay rate. The induction of LDLR expression by PPARδ agonist was further observed in liver tissue of mice and hamsters treated with L165041. Altogether, our studies identify a novel PPRE-mediated regulatory mechanism for LDLR transcription and suggest that combined treatment of statin with PPARδ agonists may have advantageous effects on LDLR expression.

Reduction of circulating PCSK9 and LDL-C levels by liver-specific knockdown of HNF1α in normolipidemic mice.

The transcription factors hepatic nuclear factor (HNF)1α and HNF1ß can bind to the HNF1 site on the proprotein convertase subtilisin/kexin type 9 (PCSK9) promoter to activate transcription in HepG2 cells. However, it is unknown whether one or both HNF1 factors are obligatory for transactivating hepatic PCSK9 gene expression in vivo. We developed shRNA adenoviral constructs (Ad-shHNF1α and Ad-shHNF1ß) to examine the effects of knockdown of HNF1α or HNF1ß on PCSK9 expression and its consequent impact on LDL receptor (LDLR) protein levels in cultured hepatic cells and liver tissue. We demonstrated that infection with Ad-shHNF1α, but not Ad-shHNF1ß, markedly reduced PCSK9 mRNA expression in HepG2 cells with a concomitant increase in LDLR protein abundance. Injecting Ad-shHNF1α in mice fed a normal diet significantly (∼ 50%) reduced liver mRNA expression and serum concentration of PCSK9 with a concomitant increase (∼ 1.9-fold) in hepatic LDLR protein abundance. Furthermore, we observed a modest but significant reduction in circulating LDL cholesterol after knockdown of HNF1α in these normolipidemic mice. Consistent with the observation that knockdown of HNF1ß did not affect PCSK9 mRNA or protein expression in cultured hepatic cells, Ad-shHNF1ß infection in mice resulted in no change in the hepatic mRNA expression or serum content of PCSK9. Altogether, our study demonstrates that HNF1α, but not HNF1ß, is the primary positive regulator of PCSK9 transcription in mouse liver.

PPARδ activation induces hepatic long-chain acyl-CoA synthetase 4 expression in vivo and in vitro.

The arachidonic acid preferred long-chain acyl-CoA synthetase 4 (ACSL4) is a key enzyme for fatty acid metabolism in various metabolic tissues. In this study, we utilized hamsters fed a normal chow diet, a high-fat diet or a high cholesterol and high fat diet (HCHFD) as animal models to explore novel transcriptional regulatory mechanisms for ACSL4 expression under hyperlipidemic conditions. Through cloning hamster ACSL4 homolog and tissue profiling ACSL4 mRNA and protein expressions we observed a selective upregulation of ACSL4 in testis and liver of HCHFD fed animals. Examination of transcriptional activators of the ACSL family revealed an increased hepatic expression of PPARδ but not PPARα in HCHFD fed hamsters. To explore a role of PPARδ in dietary cholesterol-mediated upregulation of ACSL4, we administered a PPARδ specific agonist L165041 to normolipidemic and dyslipidemic hamsters. We observed significant increases of hepatic ACSL4 mRNA and protein levels in all L165041-treated hamsters as compared to control animals. The induction of ACSL4 expression by L165041 in liver tissue in vivo was recapitulated in human primary hepatocytes and hepatocytes isolated from hamster and mouse. Moreover, employing the approach of adenovirus-mediated gene knockdown, we showed that depletion of PPARδ in hamster hepatocytes specifically reduced ACSL4 expression. Finally, utilizing HepG2 as a model system, we demonstrate that PPARδ activation leads to increased ACSL4 promoter activity, mRNA and protein expression, and consequently higher arachidonoyl-CoA synthetase activity. Taken together, we have discovered a novel PPARδ-mediated regulatory mechanism for ACSL4 expression in liver tissue and cultured hepatic cells.

Inhibition of PCSK9 transcription by berberine involves down-regulation of hepatic HNF1α protein expression through the ubiquitin-proteasome degradation pathway.

Our previous in vitro studies have identified hepatocyte nuclear factor 1α (HNF1α) as an obligated trans-activator for PCSK9 gene expression and demonstrated its functional involvement in the suppression of PCSK9 expression by berberine (BBR), a natural cholesterol-lowering compound. In this study, we investigated the mechanism underlying the inhibitory effect of BBR on HNF1α-mediated PCSK9 transcription. Administration of BBR to hyperlipidemic mice and hamsters lowered circulating PCSK9 concentrations and hepatic PCSK9 mRNA levels without affecting the gene expression of HNF1α. However, hepatic HNF1α protein levels were markedly reduced in BBR-treated animals as compared with the control. Using HepG2 cells as a model system, we obtained evidence that BBR treatment let to accelerated degradation of HNF1α protein. By applying inhibitors to selectively block the ubiquitin proteasome system (UPS) and autophagy-lysosomal pathway, we show that HNF1α protein content in HepG2 cells was not affected by bafilomycin A1 treatment, but it was dose-dependently increased by UPS inhibitors bortezomib and MG132. Bortezomib treatment elevated HNF1α and PCSK9 cellular levels with concomitant reductions of LDL receptor protein. Moreover, HNF1α protein displayed a multiubiquitination ladder pattern in cells treated with BBR or overexpressing ubiquitin. By expressing GFP-HNF1α fusion protein in cells, we observed that blocking UPS resulted in accumulation of GFP-HNF1α in cytoplasm. Importantly, we show that the BBR reducing effects on HNF1α protein and PCSK9 gene transcription can be eradicated by proteasome inhibitors. Altogether, our studies using BBR as a probe uncovered a new aspect of PCSK9 regulation by ubiquitin-induced proteasomal degradation of HNF1α.

Arachidonic acid downregulates acyl-CoA synthetase 4 expression by promoting its ubiquitination and proteasomal degradation.

ACSL4 is a member of the long-chain acyl-CoA synthetase (ACSL) family with a marked preference for arachidonic acid (AA) as its substrate. Although an association between elevated levels of ACSL4 and hepatosteatosis has been reported, the function of ACSL4 in hepatic FA metabolism and the regulation of its functional expression in the liver remain poorly defined. Here we provide evidence that AA selectively downregulates ACSL4 protein expression in hepatic cells. AA treatment decreased the half-life of ACSL4 protein in HepG2 cells by approximately 4-fold (from 17.3 ± 1.8 h to 4.2 ± 0.4 h) without causing apoptosis. The inhibitory action of AA on ACSL4 protein stability could not be prevented by rosiglitazone or inhibitors that interfere with the cellular pathways involved in AA metabolism to biologically active compounds. In contrast, treatment of cells with inhibitors specific for the proteasomal degradation pathway largely prevented the AA-induced ACSL4 degradation. We further show that ACSL4 is intrinsically ubiquitinated and that AA treatment can enhance its ubiquitination. Collectively, our studies have identified a novel substrate-induced posttranslational regulatory mechanism by which AA downregulates ACSL4 protein expression in hepatic cells.

CETP inhibitors downregulate hepatic LDL receptor and PCSK9 expression in vitro and in vivo through a SREBP2 dependent mechanism.

BACKGROUND: CETP inhibitors block the transfer of cholesteryl ester from HDL-C to VLDL-C and LDL-C, thereby raising HDL-C and lowering LDL-C. In this study, we explored the effect of CETP inhibitors on hepatic LDL receptor (LDLR) and PCSK9 expression and further elucidated the underlying regulatory mechanism. RESULTS: We first examined the effect of anacetrapib (ANA) and dalcetrapib (DAL) on LDLR and PCSK9 expression in hepatic cells in vitro. ANA exhibited a dose-dependent inhibition on both LDLR and PCSK9 expression in CETP-positive HepG2 cells and human primary hepatocytes as well as CETP-negative mouse primary hepatocytes (MPH). Moreover, the induction of LDLR protein expression by rosuvastatin in MPH was blunted by cotreatment with ANA. In both HepG2 and MPH ANA treatment reduced the amount of mature form of SREBP2 (SREBP2-M). In vivo, oral administration of ANA to dyslipidemic C57BL/6J mice at a daily dose of 50 mg/kg for 1 week elevated serum total cholesterol by approximately 24.5% (p < 0.05%) and VLDL-C by 70% (p < 0.05%) with concomitant reductions of serum PCSK9 and liver LDLR/SREBP2-M protein. Finally, we examined the in vitro effect of two other strong CETP inhibitors evacetrapib and torcetrapib on LDLR/PCSK9 expression and observed a similar inhibitory effect as ANA in a concentration range of 1-10 µM. CONCLUSION: Our study revealed an unexpected off-target effect of CETP inhibitors that reduce the mature form of SREBP2, leading to attenuated transcription of hepatic LDLR and PCSK9. This negative regulation of SREBP pathway by ANA manifested in mice where CETP activity was absent and affected serum cholesterol metabolism.

A novel posttranscriptional mechanism for dietary cholesterol-mediated suppression of liver LDL receptor expression.

It is well-established that over-accumulation of dietary cholesterol in the liver inhibits sterol-regulatory element binding protein (SREBP)-mediated LDL receptor (LDLR) gene transcription leading to a reduced hepatic LDLR mRNA level in hypercholesterolemic animals. However, it is unknown whether elevated cholesterol levels can elicit a cellular response to increase LDLR mRNA turnover to further repress LDLR expression in liver tissue. In the current study, we examined the effect of a high cholesterol diet on the hepatic expression of LDLR mRNA binding proteins in three different animal models and in cultured hepatic cells. Our results demonstrate that high cholesterol feeding specifically elevates the hepatic expression of LDLR mRNA decay promoting factor heterogeneous nuclear ribonucleoprotein (HNRNP)D without affecting expressions of other LDLR mRNA binding proteins in vivo and in vitro. Employing the approach of adenovirus-mediated gene knockdown, we further show that depletion of HNRNPD in the liver results in a marked reduction of serum LDL-cholesterol and a substantial increase in liver LDLR expression in hyperlipidemic mice. Additional studies of gene knockdown in albumin-luciferase-untranslated region (UTR) transgenic mice provide strong evidence supporting the essential role of 3'UTR in HNRNPD-mediated LDLR mRNA degradation in liver tissue. Altogether, this work identifies a novel posttranscriptional regulatory mechanism by which dietary cholesterol inhibits liver LDLR expression via inducing HNRNPD to accelerate LDLR mRNA degradation.

The critical role of mRNA destabilizing protein heterogeneous nuclear ribonucleoprotein d in 3' untranslated region-mediated decay of low-density lipoprotein receptor mRNA in liver tissue.

OBJECTIVE: Previous studies showed that low-density lipoprotein receptor (LDLR) mRNA 3' untranslated region (UTR) contains regulatory elements responsible for rapid mRNA turnover in hepatic cells and mediates the mRNA stabilization induced by berberine (BBR). Here, we elucidate the underlying mechanism of BBR's action by characterizing mRNA-binding proteins that modulate LDLR mRNA decay via 3'UTR in liver tissue in vivo. APPROACH AND RESULTS: We generated a transgenic mouse model (Alb-Luc-UTR) that expresses Luc-LDLR3'UTR reporter gene driven by the albumin promoter to study 3'UTR function in mediating LDLR mRNA decay in liver tissue. We show that treating Alb-Luc-UTR mice with BBR led to significant increases in hepatic bioluminescence signals, Luc-UTR mRNA, and LDLR mRNA levels as compared with control mice. These effects were accompanied by specific reductions of mRNA decay-promoting factor heterogeneous nuclear ribonucleoprotein D (hnRNP D) in liver of BBR-treated mice. Knockdown and overexpression studies further demonstrated that hnRNP D p37 isoform plays a major role in promoting hepatic LDLR mRNA degradation. In addition, we examined LDLR mRNA half-life, Luc-UTR reporter activity, and hnRNP D expression levels in cell lines derived from extrahepatic tissues. We demonstrated that strengths of 3'UTR in promoting mRNA degradation correlate with hnRNP D cellular abundances in nonhepatic cell lines, thereby suggesting its involvement in LDLR mRNA degradation beyond liver tissue. CONCLUSIONS: hnRNP D is critically involved in LDLR mRNA degradation in liver tissue in vivo. The inverse relationship of hnRNP D abundance with LDLR mRNA levels after BBR treatment suggests the potential of hnRNP D of being a novel therapeutic target for LDL cholesterol lowering.

Differential effects of formononetin and cladrin on osteoblast function, peak bone mass achievement and bioavailability in rats.

Dietary soy isoflavones including genistein and daidzein have been shown to have favorable effects during estrogen deficiency in experimental animals and humans. We have evaluated osteogenic effect of cladrin and formononetin, two structurally related methoxydaidzeins found in soy food and other natural sources. Cladrin, at as low as 10 nM, maximally stimulated both osteoblast proliferation and differentiation by activating MEK-Erk pathway. On the other hand, formononetin maximally stimulated osteoblast differentiation at 100 nM that involved p38 MAPK pathway but had no effect on osteoblast proliferation. Unlike daidzein, these two compounds neither activated estrogen receptor in osteoblast nor had any effect on osteoclast differentiation. Daily oral administration of each of these compounds at 10.0 mg kg(-1) day(-1) dose to recently weaned female Sprague-Dawley rats for 30 consecutive days, increased bone mineral density at various anatomic positions studied. By dynamic histomorphometry of bone, we observed that rats treated with cladrin exhibited increased mineral apposition and bone formation rates compared with control, while formononetin had no effect. Cladrin had much better plasma bioavailability compared with formononetin. None of these compounds exhibited estrogen agonistic effect in uteri. Our data suggest that cladrin is more potent among the two in promoting parameters of peak bone mass achievement, which could be attributed to its stimulatory effect on osteoblast proliferation and better bioavailability. To the best of our knowledge, this is the first attempt to elucidate structure-activity relationship between the methoxylated forms of daidzein and their osteogenic effects.

Methoxylated isoflavones, cajanin and isoformononetin, have non-estrogenic bone forming effect via differential mitogen activated protein kinase (MAPK) signaling.

Following a lead obtained from stem-bark extract of Butea monosperma, two structurally related methoxyisoflavones; cajanin and isoformononetin were studied for their effects in osteoblasts. Cajanin had strong mitogenic as well as differentiation-promoting effects on osteoblasts that involved subsequent activation of MEK-Erk and Akt pathways. On the other hand, isoformononetin exhibited potent anti-apoptotic effect in addition to promoting osteoblast differentiation that involved parallel activation of MEK-Erk and Akt pathways. Unlike genistein or daidzein, none of these two compounds appear to act via estrogen receptors in osteoblast. Once daily oral (by gavage) treatment for 30 consecutive days was given to recently weaned female Sprague-Dawley rats with each of these compounds at 10.0 mg kg(-1) day(-1) dose. Cajanin increased bone mineral density (BMD) at all skeletal sites studied, bone biomechanical strength, mineral apposition rate (MAR) and bone formation rate (BFR), compared with control. BMD levels at various anatomic positions were also increased with isoformononetin compared with control however, its effect was less potent than cajanin. Isoformononetin had no effect on the parameters of bone biomechanical strength although it enhanced MAR and BFR compared with control. Isoformononetin had very mild uterotrophic effect, whereas cajanin was devoid of any such effect. Our data suggest that cajanin is more potent than isoformononetin in accelerating peak bone mass achievement. To the best of our knowledge, this work represents the first attempt to elucidate structure-activity relationship between the two methoxylated isoflavones regarding their effects in osteoblasts and bone formation.