CAR requires Gadd45ß to promote phenobarbital-induced mouse liver tumors in early stage.

Phenobarbital (PB) is an archetypal substance used as a mouse hepatocellular carcinoma (HCC) promotor in established experimental protocols. Our previous results showed CAR is the essential factor for PB induced HCC promotion. Subsequent studies suggested Gadd45ß, which is induced by PB through CAR activation, is collaborating with CAR to repress TNF-α induced cell death. Here, we used Gadd45ß null mice (Gadd45ß KO) treated with N-diethylnitrosamine (DEN) at 5 weeks of age and kept the mice with PB supplemented drinking water from 7 to 57 weeks old. Compared with wild type mice, Gadd45ß KO mice developed no HCC in the PB treated group. Increases in liver weight were more prominent in wild type mice than KO mice. Microarray analysis of mRNA derived from mouse livers found multiple genes specifically up or down regulated in wild type mice but not null mice in DEN + PB groups. Further qPCR analysis confirmed two genes, Tgfbr2 and irisin/Fndc5, were up-regulated in PB treated wild type mice but no significant increase was observed in Gadd45ß KO mice. We focused on these two genes because previous reports showed that hepatic Irisin/Fndc5 expression was significantly higher in HCC patients and that irisin binds to TGF-ß receptor complex that includes TGFBR2 subunit. Our results revealed irisin peptide in cell culture media increased the growth rate of mouse hepatocyte-derived AML12 cells. Microarray analysis revealed that irisin-regulated genes in AML12 cells showed a significant association with the genes in the TGF-ß pathway. Expression of irisin/Fndc5 and Tgfbr2 induced growth of human HCC cell line HepG2. Thus, Gadd45ß plays an indispensable role in mouse HCC development regulating the irisin/Fndc5 and Tgfbr2 genes.

Human constitutive androstane receptor represses liver cancer development and hepatoma cell proliferation by inhibiting erythropoietin signaling.

The constitutive androstane receptor (CAR) is a nuclear receptor that plays a crucial role in regulating xenobiotic metabolism and detoxification, energy homeostasis, and cell proliferation by modulating the transcription of numerous target genes. CAR activation has been established as the mode of action by which phenobarbital-like nongenotoxic carcinogens promote liver tumor formation in rodents. This paradigm, however, appears to be unrelated to the function of human CAR (hCAR) in hepatocellular carcinoma (HCC), which remains poorly understood. Here, we show that hCAR expression is significantly lower in HCC than that in adjacent nontumor tissues and, importantly, reduced hCAR expression is associated with a worse HCC prognosis. We also show overexpression of hCAR in human hepatoma cells (HepG2 and Hep3B) profoundly suppressed cell proliferation, cell cycle progression, soft-agar colony formation, and the growth of xenografts in nude mice. RNA-Seq analysis revealed that the expression of erythropoietin (EPO), a pleiotropic growth factor, was markedly repressed by hCAR in hepatoma cells. Addition of recombinant EPO in HepG2 cells partially rescued hCAR-suppressed cell viability. Mechanistically, we showed that overexpressing hCAR repressed mitogenic EPO-EPO receptor signaling through dephosphorylation of signal transducer and activator of transcription 3, AKT, and extracellular signal-regulated kinase 1/2. Furthermore, we found that hCAR downregulates EPO expression by repressing the expression and activity of hepatocyte nuclear factor 4 alpha, a key transcription factor regulating EPO expression. Collectively, our results suggest that hCAR plays a tumor suppressive role in HCC development, which differs from that of rodent CAR and offers insight into the hCAR-hepatocyte nuclear factor 4 alpha-EPO axis in human liver tumorigenesis.

Relationship between early physical activity after total knee arthroplasty and postoperative physical function: are these related?

BACKGROUND: Physical activity is associated with physical function; however, the relationship between early physical activity after total knee arthroplasty (TKA) and postoperative physical function remains unclear. The purpose of this study was to evaluate the association of early physical activity after TKA with postoperative physical function. METHODS: Timed Up and Go test (TUG) of 47 patients was assessed preoperatively and at 10 days, 3 months, and 6 months postoperatively. Physical activity from the second to the ninth day after TKA was measured with accelerometer, and the correlation with pre- and postoperative physical function was evaluated . A multiple linear regression was used to predict TUG at 6 months after TKA. RESULTS: Postoperative physical activity correlated with preoperative TUG (ρ = -0.485, p < 0.001), TUG at 10 days (ρ = -0.675, p < 0.001), 3 months (ρ = -0.441, p < 0.01), and 6 months (ρ = -0.368, p < 0.05) after surgery. Multiple linear regression indicated that only the preoperative TUG was associated with TUG at 6 months. Postoperative physical activity was not an independent factor predicting TUG at 6 months after TKA. CONCLUSION: Our study demonstrated that patients with better physical function have higher physical activity in the early postoperative period, whereas it does not affect physical function at 6 months after TKA. In the early postoperative period, increasing physical activity may not always be necessary to improve postoperative physical function. We also confirmed that preoperative physical function affects postoperative physical function. These findings may be beneficial in improving rehabilitation programs in the early postoperative period.

Sex-specific expression mechanism of hepatic estrogen inactivating enzyme and transporters in diabetic women.

Circulating estrogens levels significantly decrease in menopause and levels off in postmenopausal women. Accordingly, the liver represses levels of enzymes and membrane transporters, thereby decreasing capability of inactivating and excreting estrogens. Women increasingly develop type 2 diabetes during or after menopause. Estrogens are known to promote liver diseases in these women. Here, we have found that the estrogen inactivating sulfotransferase (SULT1E1) and an ATP-binding cassette subfamily G member 2 (ABCG2), a gene encoding breast cancer resistance protein that exports sulfated estrogens, increased their expression levels in diabetic women but not men. For the sulfotransferase gene, phosphorylated nuclear receptors ERα and RORα, at Ser212 and Ser100, respectively, bind their response elements to activate the SULT1E1 promoter in women. This coordinated increase in estrogen inactivation and excretion, and the phosphorylated nuclear receptor-mediated gene activation could be a defense mechanism against toxicities of estrogens through inactivation and excretion in the livers of women.

Bone turnover markers in the early stage of rapidly progressive osteoarthritis of the hip.

OBJECTIVE: Previous reports have demonstrated that patients with end-stage rapidly progressive osteoarthritis of the hip (RPOH) show significantly higher serum levels of bone turnover markers than those with osteoarthritis (OA). However, the characteristics of bone turnover markers in the early stage of RPOH remain unclear. This study aimed to elucidate the association of bone turnover markers with disease progression in the early stage of RPOH. METHODS: This study included 29 postmenopausal female patients with joint space narrowing >2 mm demonstrated on a series of radiographs and computed tomography within 1 year following the onset of hip pain. The study also included 9 postmenopausal female patients with hip OA secondary to developmental dysplasia showing femoral head destruction. Cortical thickness index (CTI) associated with bone mineral density of the hip was analyzed. Serum concentrations of tartrate-resistant acid phosphatase-5b (TRACP-5b) and bone alkaline phosphatase (BAP) were evaluated. RESULTS: RPOH was classified into two types on the basis of the absence (type 1, n=13) or presence (type 2, n=16) of subsequent destruction of the femoral head within 1 year following disease onset. TRACP-5b and BAP significantly increased in RPOH type 2 compared with type 1 and OA. Receiver operating characteristic curve analyses indicated that TRACP-5b and BAP could differentiate RPOH type 2 from type 1 within 1 year following the onset. CTI showed no difference among the RPOH types 1 and 2 and OA. CONCLUSION: High serum levels of bone turnover markers may be associated with destruction of the femoral head in the early stage of RPOH.

Nuclear receptor phosphorylation in xenobiotic signal transduction.

Nuclear pregnane X receptor (PXR, NR1I2) and constitutive active/androstane receptor (CAR, NR1I3) are nuclear receptors characterized in 1998 by their capability to respond to xenobiotics and activate cytochrome P450 (CYP) genes. An anti-epileptic drug, phenobarbital (PB), activates CAR and its target CYP2B genes, whereas PXR is activated by drugs such as rifampicin and statins for the CYP3A genes. Inevitably, both nuclear receptors have been investigated as ligand-activated nuclear receptors by identifying and characterizing xenobiotics and therapeutics that directly bind CAR and/or PXR to activate them. However, PB, which does not bind CAR directly, presented an alternative research avenue for an indirect ligand-mediated nuclear receptor activation mechanism: phosphorylation-mediated signal regulation. This review summarizes phosphorylation-based mechanisms utilized by xenobiotics to elicit cell signaling. First, the review presents how PB activates CAR (and other nuclear receptors) through a conserved phosphorylation motif located between two zinc fingers within its DNA-binding domain. PB-regulated phosphorylation at this motif enables nuclear receptors to form communication networks, integrating their functions. Next, the review discusses xenobiotic-induced PXR activation in the absence of the conserved DNA-binding domain phosphorylation motif. In this case, phosphorylation occurs at a motif located within the ligand-binding domain to transduce cell signaling that regulates hepatic energy metabolism. Finally, the review delves into the implications of xenobiotic-induced signaling through phosphorylation in disease development and progression.

Characterization of rapidly progressive osteoarthritis of the hip in its early stage.

OBJECTIVE: This study aimed to characterize the process of disease progression in the early stage of rapidly progressive osteoarthritis of the hip (RPOH) and clarify its association with potential pathological factors of RPOH. METHODS: This monocentric retrospective study included 41 female patients who met the criteria for RPOH, chondrolysis >2 mm during 12 months from the onset of hip pain based on a series of radiographs and computed tomography. This study also included 9 female patients with osteoarthritis secondary to developmental dysplasia of the hip (DDH) who demonstrated chondrolysis >2 mm during 12 months from the onset of hip pain. Cortical thickness index (CTI) correlated with bone mineral density of the hip, pelvic tilt, and serum concentrations of matrix metalloproteinase (MMP)-3 were analyzed. RESULTS: RPOH was classified into two types based on the absence (type 1, n=17) and presence (type 2, n=24) of subsequent femoral head destruction within 12 months after the onset of hip pain. MMP-3 significantly increased in RPOH type 2 compared with type 1 and DDH. Increased posterior pelvic tilt was found in RPOH type 2 compared with DDH. Logistic regression and receiver operating characteristic curve analyses indicated that MMP-3 may be associated with differentiation between RPOH types 1 and 2. No difference was found in the CTI between the RPOH types and DDH. CONCLUSION: Disease progression of RPOH during 12 months after the onset of hip pain could be classified into two distinct types based on the absence (type 1) and presence (type 2) of femoral head destruction in association with MMP-3 and pelvic tilt as biological and mechanical factors, respectively. MMP-3 may be helpful to differentiate these two types in the early stage of RPOH.

Nuclear receptor CAR-ERα signaling regulates the estrogen sulfotransferase gene in the liver.

Estrogen sulfotransferase (SULT1E1) inactivates estrogen and regulates its metabolic homeostats. Whereas SULT1E1 is expressed low in the liver of adult mice, it is induced by phenobarbital (PB) treatment or spontaneously in diabetic livers via nuclear receptors. Utilizing constitutive active/androstane receptor (CAR) KO, estrogen receptor α (ERα KO, phosphorylation-blocked ERα S216A KI mice, it is now demonstrated that, after being activated by PB, CAR binds and recruits ERα onto the Sulte1 promoter for subsequent phosphorylation at Ser216. This phosphorylation tightens CAR interacting with ERα and to activates the promoter. Hepatic SULT1E1 mRNA levels are constitutively up-regulated in type 1 diabetic Akita mice; CAR spontaneously accumulates in the nucleus and activates the Sult1e1 promoter by recruiting phosphorylated ERα in the liver as observed with PB-induced livers. Thus, this CAR-phosphorylated ERα signaling enables these two nuclear receptors to communicate, activating the Sult1e1 gene in response to either PB or diabetes in mice. ERα phosphorylation may integrate CAR into estrogen actions, providing insights into understanding drug-hormone interactions in clinical therapy.

Vertebroplasty Using Allograft Bone Chips with Posterior Instrumented Fusion in the Treatment of Osteoporotic Vertebral Fractures with Neurological Deficits.

INTRODUCTION: In general, osteoporotic vertebral fractures with neurological deficits require surgery. However, the ideal surgical method remains controversial. We evaluated the efficacy of combining posterior instrumented fusion and vertebroplasty using allograft bone chips. METHODS: Twelve patients (five men, seven women; age 68-84 years, mean age 75.9 years) with osteoporotic vertebral fractures with neurological deficits were reviewed retrospectively. They underwent posterior instrumented fusion and vertebroplasty, using allograft bone, at our institution between January 2007 and June 2016. We assessed the surgical results, radiologically and neurologically, after a mean follow-up of 37.3 months. RESULTS: The mean local kyphosis angle was 10° before surgery, -3.3° immediately after surgery, and 4.4° at follow-up. The average spinal canal compromise was 26.9% before surgery and 19.5% at follow-up. All patients achieved bony fusion and none needed additional surgery. All patients improved by at least one grade on the modified Frankel grading system. CONCLUSIONS: Combining vertebroplasty, using allograft bone chips, and posterior instrumented fusion appears to be an effective option for osteoporotic vertebral fractures with neurological deficits.

A phosphorylation-deficient mutant of retinoid X receptor α at Thr 167 alters fasting response and energy metabolism in mice.

Retinoid X receptor α (RXRα) has a conserved phosphorylation motif at threonine 162 (humans) and threonine 167 (mice) within the DNA-binding domain. Here we have generated RXRα knock-in mice (RxrαT167A) bearing a single mutation of Thr 167 to alanine and examined the roles of Thr 167 in the regulation of energy metabolism within adipose, muscle, and liver tissues. RxrαT167A mice exhibited down-regulation of metabolic pathways converting glucose to fatty acids, such as acetyl-CoA carboxylase in the white adipose tissue (WAT) and ATP citrate lyase in the muscle. They also reduced gene expression for genes related to fatty acid catabolism and triglyceride synthesis in WAT and controlled heat factors such as adrenergic receptor ß1 in muscles. In contrast, hepatic gluconeogenic pathways and synthetic pathways related to fatty acids remained unaffected by this mutation. Expression of multiple genes that were affected by the Thr 167 mutation in adipose tissue exhibited clear response to LG100268, a synthetic RXR agonist. Thus, the altered gene expression in mutant mice adipose appeared to be a direct effect of RXRα Thr 167 mutation and by some secondary effect of the mutation. Blood glucose levels remained normal in RxrαT167A during feeding, as observed with RXRα wild-type mice. However, RxrαT167A mice exhibited an attenuated decrease of blood glucose levels that occurred after fasting. This attenuation correlated with a concomitant down-regulation of lipid metabolism in WAT and was associated with RXRα phosphorylation at Thr 167. Thus, Thr 167 enabled RXRα to coordinate these three organs for regulation of energy metabolism and maintenance of glucose homeostasis.

Phosphorylated Nuclear Receptor CAR Forms a Homodimer To Repress Its Constitutive Activity for Ligand Activation.

The nuclear receptor CAR (NR1I3) regulates hepatic drug and energy metabolism as well as cell fate. Its activation can be a critical factor in drug-induced toxicity and the development of diseases, including diabetes and tumors. CAR inactivates its constitutive activity by phosphorylation at threonine 38. Utilizing receptor for protein kinase 1 (RACK1) as the regulatory subunit, protein phosphatase 2A (PP2A) dephosphorylates threonine 38 to activate CAR. Here we demonstrate that CAR undergoes homodimer-monomer conversion to regulate this dephosphorylation. By coexpression of two differently tagged CAR proteins in Huh-7 cells, mouse primary hepatocytes, and mouse livers, coimmunoprecipitation and two-dimensional gel electrophoresis revealed that CAR can form a homodimer in a configuration in which the PP2A/RACK1 binding site is buried within its dimer interface. Epidermal growth factor (EGF) was found to stimulate CAR homodimerization, thus constraining CAR in its inactive form. The agonistic ligand CITCO binds directly to the CAR homodimer and dissociates phosphorylated CAR into its monomers, exposing the PP2A/RACK1 binding site for dephosphorylation. Phenobarbital, which is not a CAR ligand, binds the EGF receptor, reversing the EGF signal to monomerize CAR for its indirect activation. Thus, the homodimer-monomer conversion is the underlying molecular mechanism that regulates CAR activation, by placing phosphorylated threonine 38 as the common target for both direct and indirect activation of CAR.

Seasonal variation in adult hip disease secondary to osteoarthritis and developmental dysplasia of the hip.

AIM: To determine if there was a seasonal variation in adults undergoing total hip arthroplasty for end stage hip disease due to osteoarthritis (OA) or sequelae of developmental dysplasia of the hip (DDH). METHODS: The total hip registry from the author's institution for the years 1969 to 2013 was reviewed. The month of birth, age, gender, and ethnicity was recorded. Differences between number of births observed and expected in the winter months (October through February) and non-winter mo (March through September) were analyzed with the χ2 test. Detailed temporal variation was mathematically assessed using cosinor analysis. RESULTS: There were 7792 OA patients and 60 DDH patients who underwent total hip arthroplasty. There were more births than expected in the winter months for both the DDH (P < 0.0001) and OA (P = 0.0052) groups. Cosinor analyses demonstrated a peak date of birth on 1st October. CONCLUSION: These data demonstrate an increased prevalence of DDH and OA in those patients born in winter.

Clinical predictors for possible failure after total hip arthroplasty.

INTRODUCTION: With the rising number of total hip arthroplasties (THAs) each year, it is increasingly important for surgeons to have evidence-based information on which to determine how often patients should be examined postoperatively. The purpose of this research was to determine whether it is possible to identify - based on Harris Hip Score (HHS) - early signs or predictors of THA failure so that methods of postoperative follow-up can be scheduled in advance of the time frame indicated by those predictors of failure. METHODS: The HHS of 9,949 primary THAs performed from 1973 to 2012 was reviewed retrospectively to identify the clinical predictors of failure. 1,131 hips were completely lost to follow-up, leaving 8,331 primary THAs in 6,979 patients. Time to failure was recorded with Kaplan-Meier analysis performed with aseptic loosening or revision of any component as the endpoint. RESULTS: Regression analysis revealed that a pain score of 30 or less at any time of follow-up (p<0.0001) was a significant risk and strongly indicative of later failing. A low distance walked score of 5 or less at 6 months (p = 0.0087) and 1 year (p = 0.0167) served as an early predictor of future failure. A lower stairs score of 2 or less was also an early predictor at 1 year (p = 0.0343) and at 3 years (p = 0.0245). A lower limp score of 8 or less was a mid-term predictor at 3 (p = 0.0001), 5 (p = 0.0002), 7 (p = 0.0191) and 10 (0.0028) years postoperative follow-up. CONCLUSIONS: Pain, walk, stairs and limp scores are predictive of THA failure. Surgeons with patients who present with these indicators should optimise postoperative follow-ups to alert their patients.

Twenty-Five-Years and Greater, Results After Nonmodular Cemented Total Knee Arthroplasty.

BACKGROUND: Total knee arthroplasty (TKA) has been shown to be very successful with long-term follow-ups. But there are no reports showing prosthesis survival at 25-30 years. Here, we report the outcomes for 25-30 years using the Anatomic Graduated Component (Biomet, Warsaw, IN) TKA and elucidate the etiology and cause of failure of the components. METHODS: We reviewed the outcomes of 5649 primary total knee arthroplasties for 25-30 years using the Anatomic Graduated Component. Statistical analysis was performed by the Kaplan-Meier survival analysis. Clinical outcomes included the Knee Society Score and standardized radiographs to check for loosening of the implants. The reason for revision surgery was reviewed retrospectively. We compared our results with those at another institution with similar long-term follow-up. RESULTS: There were 112 failures, 48 with aseptic loosening and 25 with instability for an overall prosthesis survival rate of 94.2% at 25 years and 92.4% at 30 years follow-up. In the third decade after TKA, patients are substantially more likely to experience death than experience a failing prosthesis, with a 3811% greater risk of dying relative to failing (Risk ratio = 38.1, Odds ratio = 56.7, P < .0001). CONCLUSION: There was a greater risk of dying than failing over time. The primary reason for revision knee surgery was due to aseptic loosening of the prosthesis followed by instability.

Short-term Outcomes With a Second-Generation Uncemented Stem in Total Hip Arthroplasty.

An uncemented stem has been used successfully in total hip arthroplasty for 2 decades, and some implants have been updated. The authors have used second-generation uncemented proximal porous coating stems, the Echo Bi-Metric Full Proximal Profile stem (Echo FPP; Biomet, Warsaw, Indiana) and the Echo Reduced Proximal Profile stem (Echo RPP; Biomet). This article reports short-term outcomes with these stems compared with their predecessor, the Bi-Metric stem (Biomet). The authors reviewed 1280 Echo FPP stems, 366 Echo RPP stems, and 1497 Bi-Metric stems. With more than 5 years of follow-up, both the Echo FPP and the Echo RPP stems had 100% survivorship with stem revision as the endpoint, and the Bi-Metric stems also had 100% survivorship for more than 20 years. Average Harris Hip Scores for the Echo FPP, the Echo RPP, and the Bi-Metric stems were 53.3, 49.7, and 51.5 preoperatively, 93.8, 94.6, and 95.2 at 1 year, and 94.0, 95.8, and 95.4 at 3 years postoperatively, respectively. Dislocation after surgery was significantly lower with the Echo FPP and the Echo RPP stems than with the Bi-Metric stems, but this was more relevant to the surgical approach and head size. All radiographs of the Echo FPP, the Echo RPP, and the Bi-Metric stems showed proximal femoral remodeling consistent with osseous ingrowth. Distal cortical hypertrophy around the implanted stem and spot-welding were comparably observed in all 3 cohorts. The short-term outcomes of the updated uncemented stems were as excellent as the previously used stem regarding survivorship, complications, and radiographic assessment.

SLC13A5 is a novel transcriptional target of the pregnane X receptor and sensitizes drug-induced steatosis in human liver.

The solute carrier family 13 member 5 (SLC13A5) is a sodium-coupled transporter that mediates cellular uptake of citrate, which plays important roles in the synthesis of fatty acids and cholesterol. Recently, the pregnane X receptor (PXR, NR1I2), initially characterized as a xenobiotic sensor, has been functionally linked to the regulation of various physiologic processes that are associated with lipid metabolism and energy homeostasis. Here, we show that the SLC13A5 gene is a novel transcriptional target of PXR, and altered expression of SLC13A5 affects lipid accumulation in human liver cells. The prototypical PXR activator rifampicin markedly induced the mRNA and protein expression of SLC13A5 in human primary hepatocytes. Utilizing cell-based luciferase reporter assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays, we identified and functionally characterized two enhancer modules located upstream of the SLC13A5 gene transcription start site that are associated with regulation of PXR-mediated SLC13A5 induction. Functional analysis further revealed that rifampicin can enhance lipid accumulation in human primary hepatocytes, and knockdown of SLC13A5 expression alone leads to a significant decrease of the lipid content in HepG2 cells. Overall, our results uncover SLC13A5 as a novel target gene of PXR and may contribute to drug-induced steatosis and metabolic disorders in humans.

Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.

Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr(52), which then promoted the dephosphorylation of CAR at Thr(38) by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.

Conditional deletion of Tgfbr2 in hypertrophic chondrocytes delays terminal chondrocyte differentiation.

Transforming growth factor ß (Tgfb) signaling plays an important role in endochondral ossification. Previous studies of mice in which the Tgfb type II receptor gene (Tgfbr2) was deleted in the limb bud mesenchymal cells or differentiated chondrocytes showed defects in the development of the long bones or the axial skeleton, respectively. Here, we generated mouse embryos in which the Tgfbr2 gene was ablated in hypertrophic chondrocytes. These mice exhibited delays in both the hypertrophic conversion of proliferating chondrocytes and the subsequent terminal chondrocyte differentiation. The expression domains of Col10a1, Matrix metalloproteinase 13, and Osteopontin were small, and the expression of Vascular endothelial growth factor and Platelet endothelial cell adhesion molecule was downregulated. The calcification of the bone collar in the mutant mice was markedly delayed and the periosteum was thin, possibly because of the downregulation of Indian hedgehog expression. We conclude that Tgfb signaling in hypertrophic chondrocytes positively regulates terminal chondrocyte differentiation, angiogenesis in calcified cartilage, and osteogenesis in the bone collar, at least partly through Indian hedgehog signaling in vivo.

Garlic extract diallyl sulfide (DAS) activates nuclear receptor CAR to induce the Sult1e1 gene in mouse liver.

Constituent chemicals in garlic extract are known to induce phase I and phase II enzymes in rodent livers. Here we have utilized Car(+/+) and Car(-/-) mice to demonstrate that the nuclear xenobiotic receptor CAR regulated the induction of the estrogen sulfotransferase Sult1e1 gene by diallyl sulfide (DAS) treatment in mouse liver. DAS treatment caused CAR accumulation in the nucleus, resulting in a remarkable increase of SULT1E1 mRNA (3,200 fold) and protein in the livers of Car(+/+) females but not of Car(-/-) female mice. DAS also induced other CAR-regulated genes such as Cyp2b10, Cyp3a11 and Gadd45ß. Compared with the rapid increase of these mRNA levels, which began as early as 6 hours after DAS treatment, the levels of SULT1E1 mRNA began increasing after 24 hours. This slow response to DAS suggested that CAR required an additional factor to activate the Sult1e1 gene or that this activation was indirect. Despite the remarkable induction of SULT1E1, there was no decrease in the serum levels of endogenous E2 or increase of estrone sulfate while the clearance of exogenously administrated E2 was accelerated in DAS treated mice.

Comparative study of human and mouse pregnane X receptor agonistic activity in 200 pesticides using in vitro reporter gene assays.

The nuclear receptor, pregnane X receptor (PXR), is a ligand-dependent transcription factor that regulates genes involved in xenobiotic metabolism. Recent studies have shown that PXR activation may affect energy metabolism as well as the endocrine and immune systems. In this study, we characterized and compared the agonistic activities of a variety of pesticides against human PXR (hPXR) and mouse PXR (mPXR). We tested the hPXR and mPXR agonistic activity of 200 pesticides (29 organochlorines, 11 diphenyl ethers, 56 organophosphorus pesticides, 12 pyrethroids, 22 carbamates, 12 acid amides, 7 triazines, 7 ureas, and 44 others) by reporter gene assays using COS-7 simian kidney cells. Of the 200 pesticides tested, 106 and 93 activated hPXR and mPXR, respectively, and a total of 111 had hPXR and/or mPXR agonistic activity with greater or lesser inter-species differences. Although all of the pyrethroids and most of the organochlorines and acid amides acted as PXR agonists, a wide range of pesticides with diverse structures also showed hPXR and/or mPXR agonistic activity. Among the 200 pesticides, pyributicarb, pretilachlor, piperophos and butamifos for hPXR, and phosalone, prochloraz, pendimethalin, and butamifos for mPXR, acted as particularly potent activators at low concentrations in the order of 10⁻8-10⁻7 M. In addition, we found that several organophosphorus oxon- and pyributicarb oxon-metabolites decreased PXR activation potency compared to their parent compounds. These results suggest that a large number of structurally diverse pesticides and their metabolites possess PXR-mediated transcriptional activity, and their ability to do so varies in a species-dependent manner in humans and mice.