Effects of Bisphenol A on expression of genes related to amino acid transporters, insulin- like growth factor, aquaporin and amino acid release by porcine trophectoderm cells.

The peri-implantation period of pregnancy is critical for conceptus development, implantation, and signaling for establishment of pregnancy. This study evaluated the effects of bisphenol A (BPA) on proliferation, adhesion, and migration of porcine trophectoderm (pTr2) cells, expression of transporters of arginine and synthesis of amino acids. All concentrations of BPA decreased proliferation and adhesion of pTr2 cells after 96 h compared to the control group. Lower concentrations of BPA (1 × 10-9, 1 × 10-8, 10-7M) increased (P < 0.05), but higher concentrations of BPA (1 × 10-5, 1 × 10-4 M) decreased migration of pTr2 cells. BPA increased expression of SLC7A1 mRNA at lower concentrations (1 × 10-9 to 1 × 10-6M) and SL7A6, another cationic acid transporter, at higher concentrations (1 × 10-5, 1 × 10-4 M). BPA also down-regulated the expression of IGF1 and IGF1 receptor at concentrations of 1 × 10-7 to 1 × 10-4 M compared to the control group. The expression of mRNAs for aquaporins (AQP) 3 and 4 were reduced at all concentrations of BPA, but at lower concentrations of BPA, (1 × 10-9 to 1 × 10-8M) expression of AQP9 mRNA increased and the expression of AQP11 was not affected by BPA (P > 0.05). There was an inhibitory effect of BPA on the release of synthesis of asparagine, threonine, taurine, tryptophan, and ornithine into the culture medium by pTr2 cells. Collectively, BPA adversely affected the expression of transporters for cationic amino acids like arginine, as well as AQPs, IGF1, and IGF1R associated with proliferation, migration, and adhesion of pTr2 cells. Those adverse effects would likely increase pregnancy losses during the peri-implantation period of pregnancy.

Effects of BPA on expression of apoptotic genes and migration of ovine trophectoderm (oTr1) cells during the peri-implantation period of pregnancy.

Bisphenol A (BPA) is an endocrine-disrupting chemical used in the manufacture of many products used daily. In the present study, the effects of BPA (1 × 10-4 to 1 × 10-9 M) on migration and on the expression of some apoptotic genes were examined in vitro using ovine trophectoderm (oTr1) primary cell line. The results revealed that BPA at 1 × 10-9, 1 × 10-8 and 1 × 10-7M increased migration of oTr1 cells, while 1 × 10-6, 1 × 10-5 and 1 × 10-4 M BPA decreased cell migration. Regarding apoptosis, expression of the anti-apoptotic gene Bcl-2 mRNA was greater at 1 × 10-8 and 1 × 10-9 M BPA and was down-regulated at 1 × 10-4 to 1 × 10-7 M BPA; however, expression of pro-apoptotic genes (Bax, cathepsin B, caspase-3 and c-myc) was reduced at the higher concentrations of BPA. Results of this study suggest that BPA may impair implantation by decreasing migration of oTr1 cells and inhibiting apoptosis.

Effects of catecholamines on secretion of interferon tau and expression of genes for synthesis of polyamines and apoptosis by ovine trophectoderm.

Ovine trophectoderm (oTr1) cells were used to investigate effects of epinephrine (EP), norepinephrine (NE), and dopamine (DA) on their proliferation, migration and adhesion, secretion of interferon tau (IFNT), and expression of genes for synthesis of polyamines and apoptosis. Expression of mRNAs for agmatinase (AGMAT), arginine decarboxylase (ADC), ornithine decarboxylase (ODC1), and solute carrier family 7 (SLC7A1) (cationic amino acid transporter, Y + system), member 1 increased (P < 0.05) in oTr1 cells in response to EP and DA. However, expression of SLC7A1 decreased at high doses of EP and expression of ADC mRNA by oTr1 cells decreased in response to 20 and 40 ng/ml NE, and 40 ng/ml DA. Migration of oTr1 cells increased in response to EP, DA, and NE after 48 h of treatment. However, proliferation of oTr1 cells was inhibited by 300 pg/ml EP after 96 h and DA at 20 and 100 ng/ml. EP increased adhesion of oTr1 cells. The secretion of IFNT increased in response to 300 pg/ml EP, 100 ng/ml NE and DA after 48 h and at 96 h, and both DA (40 ng/ml) and NE (100 ng/ml). Expression of mRNAs for apoptotic genes (caspase 3, cathpsin B, BCL2 associated X protein "bax," B-cell lymphoma 2 "bcl2," and proto-oncogene "cmyc") decreased (P < 0.05) in response to catecholamines, but DA did not affect (P < 0.05) expression of cMYC mRNA. These results indicate that catecholamines play important roles in conceptus development during the peri-implantation period of pregnancy through effects on synthesis of polyamines, secretion of IFNT, and expression of apoptotic genes by oTr1 cells.

Effects of Bisphenol-A on proliferation and expression of genes related to synthesis of polyamines, interferon tau and insulin-like growth factor 2 by ovine trophectoderm cells.

This study evaluated the effects of bisphenol A (BPA) on proliferation of ovine trophectoderm (oTr1) cells, as well as expression of genes for transport of arginine and synthesis of polyamines. BPA reduced proliferation of oTr1 cells at concentrations of 1 × 10-6, 1 × 10-5, 1 × 10-4 M compared to concentrations of 0, 1 × 10-9, and 1 × 10-8 M at 24 and 96 h of culture. Lower concentrations of BPA significantly increased expression of mRNAs for agmatinase (AGMAT), arginine decarboxylase (ADC), ornithine decarboxylase (ODC1) and solute carrier family 7 member 1 (SLC7A1). Similarly, synthesis of polyamines by oTr1 cells was greatest at lower concentrations of BPA and decreased as the dose of BPA increased. Expression of mRNAs for interferon tau (IFNT) and insulin-like growth factor 2 (IGF2) by oTr1 cells was greater than for controls at 1 × 10-9 M BPA. Overall, the effects of BPA on proliferation and gene expression by oTr1 cells were highly dose-dependent.

Functional roles of agmatinase during the peri-implantation period of pregnancy in sheep.

This study investigated the effect of agmatine (Agm) in proliferation of ovine trophecdoderm cells (oTr1) as well as the importance of the arginine decarboxylase (ADC) and agmatinase (AGMAT) alternative pathway for synthesis of polyamines in ovine conceptuses during the peri-implantation period of pregnancy. Morpholino antisense oligonucleotides (MAOs) were used to inhibit translation of mRNAs for ODC1 alone, AGMAT alone, and their combination. Rambouillet ewes (N = 50) were assigned randomly to the following treatments on Day 8 of pregnancy: MAO control (n = 10); MAO-ODC1 (n = 8); MAO-ADC (n = 6); MAO-ODC1:MAO-ADC (n = 9); or MAO-ODC1:MAO-AGMAT (n = 9). Ewes were ovario-hysterectomized on Day 16 of pregnancy to obtain uterine flushings, uterine endometrium, and conceptus tissues. Inhibition of translation of both ODC1 and AGMAT resulted in 22% of ewes having morphologically and functionally normal (elongated and healthy) conceptuses designated MAO-ODC1:MAO-AGMAT (A). But, 78% of the MAO-ODC1:MAO-AGMAT ewes had morphologically and functionally abnormal (not elongated and fragmented) conceptuses designated MAO-ODC1:MAO-AGMAT (B). The pregnancy rate was less (22%; P < 0.05) for MAO-ODC1:MAO-AGMAT ewes than for MAO-control (80%), MAO-ODC1 (75%), MAO-ADC (84%), and MAO-ODC1:MAO-ADC (44%) ewes. Moreover, inhibition of translational of both ODC1 and AGMAT mRNAs increased expression of ADC, SLC22A1, SLC22A2, and SLC22A3 mRNAs, as well as abundances of agmatine, putrescine, spermindine, and spermine in conceptus tissue. However, MAO-ODC1:AGMAT(B) ewes had greater abundances of agmatine, putrescine, and spermidine and reduced amounts of spermine in uterine flushes. Thus, in vivo knockdown of translation of ODC1 and AGMAT mRNAs increased expression of genes for the synthesis and transport of polyamines in ovine conceptuses during the peri-implantation period of pregnancy.

Effects of agmatine on secretion of interferon tau and catecholamines and expression of genes related to production of polyamines by ovine trophectoderm cells.

Embryonic survival requires histotrophic nutrition, including molecules secreted or transported into the uterine lumen by uterine epithelia. L-Arginine (Arg) is a common substrate for synthesis of nitric oxide, ornithine, proline, glutamate, creatinine, urea, polyamines and agmatine. Agmatine (Agm) is a product of arginine decarboxylation and it is a substrate for agmatinase for synthesis of putrescine and other polyamines in the ovine conceptus. Polyamines are essential for conceptus development. Therefore, this study compared effects of Arg and Agm on the behavior of ovine trophectoderm (oTr1) cells cultured in vitro. Arg, but not Agm, increased proliferation and migration of oTr1 cells, but neither Arg nor Agm affected cell adhesion. The total amount of IFNT in culture medium of oTr1 cells was increased by Arg, but Agm increased the IFNT production per oTr1 cell. Arg and Agm plus Arg decreased secretion of dopamine and norepinephrine by oTr1 cells. Agm upregulates expression of mRNAs SLC7A1, agmatinase and OAZ2 while the combination of Arg and Agm decreased expression of mRNAs for ODC1, SLC7A1, OAZ1 and OAZ3 by oTr1 cells. Although Agm does not stimulate proliferation, migration or adhesion of oTr1 cells or their secretion of catecholamines, Agm did increase transcription of SLC7A1, agmatinase and OAZ2 genes which would increase the capacity of oTr1 cells to produce polyamines. Collectively, our findings suggest a role for Arg and Agm in the regulation of transport of basic amino acids (including Arg), polyamine synthesis, and secretion of catecholamines by oTr1 cells.

Preparation of temperature sensitive molecularly imprinted polymer for solid-phase microextraction coatings on stainless steel fiber to measure ofloxacin.

A kind of new temperature sensitive molecularly imprinted polymer (MIP) with ofloxacin (OFL) as template was prepared for the coating of solid phase microextraction (SPME). Dopamine was self-polymerized on stainless steel fiber (SSF) as the SPME support followed by silanization. Then MIP was synthesized as SPME coating on the modified SSF in a capillary, with N-isopropyl acrylamide as temperature sensitive monomer and methacrylic acid as functional monomer. The synthesis could be well repeated with multiple capillaries putting in the same reaction solution. The obtained MIP fiber was evaluated in detail with different techniques and various adsorption experiments. At last the MIP fiber was used to extract the OFL in milk. Satisfied recoveries between 89.7 and 103.4% were obtained with the limit of quantification (LOQLC) of 0.04 µg mL(-1) by the method of SPME coupled with high performance of liquid chromatography (HPLC).

Synthesis and characterization of biomimetic citrate-based biodegradable composites.

Natural bone apatite crystals, which mediate the development and regulate the load-bearing function of bone, have recently been associated with strongly bound citrate molecules. However, such understanding has not been translated into bone biomaterial design and osteoblast cell culture. In this work, we have developed a new class of biodegradable, mechanically strong, and biocompatible citrate-based polymer blends (CBPBs), which offer enhanced hydroxyapatite binding to produce more biomimetic composites (CBPBHAs) for orthopedic applications. CBPBHAs consist of the newly developed osteoconductive citrate-presenting biodegradable polymers, crosslinked urethane-doped polyester and poly (octanediol citrate), which can be composited with up to 65 wt % hydroxyapatite. CBPBHA networks produced materials with a compressive strength of 116.23 ± 5.37 MPa comparable to human cortical bone (100-230 MPa), and increased C2C12 osterix gene and alkaline phosphatase gene expression in vitro. The promising results above prompted an investigation on the role of citrate supplementation in culture medium for osteoblast culture, which showed that exogenous citrate supplemented into media accelerated the in vitro phenotype progression of MG-63 osteoblasts. After 6 weeks of implantation in a rabbit lateral femoral condyle defect model, CBPBHA composites elicited minimal fibrous tissue encapsulation and were well integrated with the surrounding bone tissues. The development of citrate-presenting CBPBHA biomaterials and preliminary studies revealing the effects of free exogenous citrate on osteoblast culture shows the potential of citrate biomaterials to bridge the gap in orthopedic biomaterial design and osteoblast cell culture in that the role of citrate molecules has previously been overlooked.

Preparation, characterization, and application of a new stationary phase containing different kinds of amine groups.

A new stationary phase containing primary amine and tertiary amine groups was synthesized and its structure was confirmed by infrared spectra, elemental analysis, and zeta potential. This new material was packed into a stainless steel column and the effect of acetonitrile (ACN) content in mobile phase on retention was evaluated using probes of nucleosides, sulfanilamides, and quinolone compounds. This showed hydrophilic interaction, ion interaction, or a mixed-mode retention mechanism for different kinds of solutes, respectively. The effects of the pH and ion strength of the mobile phase were investigated to further understand the retention mechanism of the new stationary phase. The interaction forces caused by the new phase included adsorption, electrostatic function, and ion exchange. The new material could be used as a stationary phase in different high-performance liquid chromatography modes.

Lactoferrin activates BMP7 gene expression through the mitogen-activated protein kinase ERK pathway in articular cartilage.

Rheumatoid arthritis (RA) is a chronic inflammatory disease leading to destruction of cartilage in joints. Neutrophil granulocytes are the predominant cell type in the synovial fluid of affected joints. Neutrophils release stimulants that alter the chondrocyte metabolism. Lactoferrin (LF) is a marker of neutrophil granulocyte activation. Local concentrations of LF in synovial fluid are much higher in the joints of RA patients. However, the effect of LF on articular cartilage of joints is not well understood. Effect of LF on gene expression in primary chondrocytes of articular cartilage was investigated in this study. We found that LF preferentially activated BMP7 expression rather than BMP2 or BMP4, and that LF activated BMP7 expression in a dose-dependent and time-dependent manner. Interestingly, a specific mitogen-activated protein kinase ERK inhibitor U0126, but not JNK kinase inhibitor SP600125, abrogated LF activation of BMP7 gene expression. LF-induced increase in BMP7 protein level was in parallel with the phosphorylation of ERK in primary chondrocytes. Taken together, we provide the first evidence to demonstrate that LF activates BMP7 expression through the mitogen-activated protein kinase ERK pathway in primary chondrocytes of articular cartilage. Since BMP7 is important for the maintenance of homeostasis in articular cartilage, we speculate that there is a protective function of LF at the site of joint inflammation.

Matrix metalloproteinase 13 (MMP13) is a direct target of osteoblast-specific transcription factor osterix (Osx) in osteoblasts.

Osterix (Osx) is an osteoblast-specific transcription factor required for bone formation and osteoblast differentiation from mesenchymal stem cells. In Osx-null mice, no bone formation occurs. Matrix metalloproteinase 13 (MMP13) is a member of the matrix metalloproteinase family and plays an important role in endochondral ossification and bone remodeling. Transcriptional regulation of MMP13 expression in osteoblasts is not well understood. Here, we provide several lines of evidence which show that MMP13 is a direct target of Osx in osteoblasts. Calvaria obtained from Osx-null embryos displayed dramatic reductions in MMP13 expression compared to wild-type calvaria. Stable overexpression of Osx stimulated MMP13 expression in C2C12 mesenchymal cells. Inhibition of Osx expression by siRNA led to downregulation of MMP13 expression. Mechanistic approaches using transient transfection assays showed that Osx directly activated a 1 kb fragment of the MMP13 promoter in a dose-dependent manner. To define the region of the MMP13 promoter that was responsive to Osx, a series of MMP13 promoter deletion mutants were examined and the minimal Osx-responsive region was refined to the proximal 80 bp of the MMP13 promoter. Additional point mutant analysis was used to identify one GC-rich region that was responsible for MMP13 promoter activation by Osx. Gel Shift Assay showed that Osx bound to MMP13 promoter sequence directly. Chromatin immunoprecipitation assays demonstrated that endogenous Osx was associated with the native MMP13 promoter in primary osteoblasts in vivo. Taken together, these data strongly support a direct regulatory role for Osx in MMP13 gene expression in osteoblasts. They further provide new insight into potential mechanisms and pathways that Osx controls bone formation.

Osteoblast-specific transcription factor Osterix (Osx) and HIF-1α cooperatively regulate gene expression of vascular endothelial growth factor (VEGF).

Bone formation is a highly regulated process involving the differentiation of mesenchymal stem cells to osteoblasts. Angiogenesis and osteogenesis are tightly coupled during bone formation. Vascular endothelial growth factor (VEGF) is involved in both processes. Relatively little is known about VEGF gene regulation in osteoblasts. Osterix (Osx) is a bone morphogenetic protein 2 (BMP-2) inducible osteoblast-specific transcription factor required for osteoblast differentiation and bone formation. Our recent study has demonstrated that Osx controls VEGF expression in osteoblasts. Here, we further characterized Osx regulation of VEGF. To address which domain of Osx is responsible for VEGF regulation, the deletion mutant analysis and transfection assay were carried out to show that proline-rich region (PRR) is required for Osx activation of VEGF promoter activity. Hypoxia-inducible factor-1α (HIF-1α) has been reported to couple angiogenesis to osteogenesis, and to upregulate VEGF. Effect of Osx on HIF-1α expression was examined in this study. Quantitative RT-PCR results revealed that HIF-1α remained unchanged between wild type and Osx knockout calvaria at E18.5 in mouse embryos. Overexpression of Osx in stable C2C12 mesenchymal cells using Tet-off system did not affect HIF-1α expression. HIF-1α level did not change after Osx inhibition by siRNA in osteoblasts. Moreover, BMP-2 stimulation led to upregulation of Osx and VEGF, but not HIF-1α. These results demonstrate that HIF-1α is not a downstream target of Osx in osteoblasts, suggesting that Osx regulation of VEGF is independent of HIF-1α expression level. Interestingly, synergistic interplays were observed between Osx and HIF-1α in VEGF promoter activation in transfection assay. Our findings indicate that Osx and HIF-1α cooperatively regulate VEGF expression.

Transcriptional regulation of Vascular Endothelial Growth Factor (VEGF) by osteoblast-specific transcription factor Osterix (Osx) in osteoblasts.

Osterix (Osx) is an osteoblast-specific transcription factor required for bone formation and osteoblast differentiation. The critical step in bone formation is to replace the avascular cartilage template with vascularized bone. Osteogenesis and angiogenesis are associated with each other, sharing some essential regulators. Vascular endothelial growth factor (VEGF) is involved in both angiogenesis and osteogenesis. Transcriptional regulation of VEGF expression is not well known in osteoblasts. In this study, quantitative real-time RT-PCR results revealed that VEGF expression was down-regulated in Osx-null calvarial cells and that osteoblast marker osteocalcin expression was absent. Overexpression of Osx in stable C2C12 mesenchymal cells using a Tet-off system resulted in up-regulation of both osteocalcin and VEGF expression. The inhibition of Osx by siRNA led to repression of VEGF expression in osteoblasts. These results suggest that Osx controls VEGF expression. Transfection assays demonstrated that Osx activated VEGF promoter activity. A series of VEGF promoter deletion mutants were examined and the minimal Osx-responsive region was defined to the proximal 140-bp region of the VEGF promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VEGF promoter activation by Osx. Gel shift assay showed that Osx bound to the VEGF promoter sequence directly. Chromatin immunoprecipitation assays indicated that endogenous Osx associated with the native VEGF promoter in primary osteoblasts. Moreover, immunohistochemistry staining showed decreased VEGF protein levels in the tibiae of Osx conditional knock-out mice. We provide the first evidence that Osx controlled VEGF expression, suggesting a potential role of Osx in coordinating osteogenesis and angiogenesis.

Osteoblast-specific transcription factor Osterix increases vitamin D receptor gene expression in osteoblasts.

Osterix (Osx) is an osteoblast-specific transcription factor required for osteoblast differentiation from mesenchymal stem cells. In Osx knock-out mice, no bone formation occurs. The vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily that regulates target gene transcription to ensure appropriate control of calcium homeostasis and bone development. Here, we provide several lines of evidence that show that the VDR gene is a target for transcriptional regulation by Osx in osteoblasts. For example, calvaria obtained from Osx-null embryos displayed dramatic reductions in VDR expression compared to wild-type calvaria. Stable overexpression of Osx stimulated VDR expression in C2C12 mesenchymal cells. Inhibition of Osx expression by siRNA led to downregulation of VDR. In contrast, Osx levels remained unchanged in osteoblasts in VDR-null mice. Mechanistic approaches using transient transfection assays showed that Osx directly activated a 1 kb fragment of the VDR promoter in a dose-dependent manner. To define the region of the VDR promoter that was responsive to Osx, a series of VDR promoter deletion mutants were examined and the minimal Osx-responsive region was refined to the proximal 120 bp of the VDR promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VDR promoter activation by Osx. Chromatin immunoprecipitation assays demonstrated that endogenous Osx was associated with the native VDR promoter in primary osteoblasts in vivo. Cumulatively, these data strongly support a direct regulatory role for Osx in VDR gene expression. They further provide new insight into potential mechanisms and pathways that Osx controls in osteoblasts and during the process of osteoblastic cell differentiation.

Osteoblast-specific transcription factor Osterix (Osx) is an upstream regulator of Satb2 during bone formation.

Osterix (Osx) is an osteoblast-specific transcription factor essential for osteoblast differentiation and bone formation. Osx knock-out mice lack bone completely. Satb2 is critical for osteoblast differentiation as a special AT-rich binding transcription factor. It is not known how Satb2 is transcriptionally regulated during bone formation. In this study, quantitative real-time RT-PCR results demonstrated that Satb2 was down-regulated in Osx-null calvaria. In stable C2C12 mesenchymal cells using the tetracycline (Tet)-Off system, overexpression of Osx stimulated Satb2 expression. Moreover, inhibition of Osx by siRNA led to repression of Satb2 expression in osteoblasts. These results suggest that Osx controls Satb2 expression. Transient transfection assay showed that Osx activated 1kb Satb2 promoter reporter activity in a dose-dependent manner. To define the region of Satb2 promoter responsive to Osx activation, a series of deletion mutants of Satb2 constructs were made, and the minimal region was narrowed down to the proximal 130 bp of the Satb2 promoter. Further point mutation studies found that two GC-rich region mutations disrupted the Satb2 130bp promoter activation by Osx, suggesting that these GC-rich binding sites were responsible for Satb2 activation by Osx. Gel shift assay showed that Osx bound to the Satb2 promoter sequence directly. ChIP assays indicated that endogenous Osx associated with the native Satb2 promoter in osteoblasts. Importantly, Satb2 siRNA significantly inhibited Osx-induced osteoblast marker gene expressions. Taken together, our findings indicate that Osx is an upstream regulator of Satb2 during bone formation. This reveals a new additional link of the transcriptional regulation mechanism that Osx controls bone formation.

Glycopolypeptide-encapsulated Mn-doped ZnS quantum dots for drug delivery: fabrication, characterization, and in vitro assessment.

In this work, glycopolypeptide nanoparticles (GPNPs) composed of Mn doped ZnS quantum dots (QDs) as core and glycopolypeptides as shell were synthesized through the surface initiated polymerization of N-carboxyanhydrides and surface condensation polymerization with carboxymethyldextran on amine-functionalized QDs. The loading and release behavior of ibuprofen on the GPNPs were investigated. The GPNPs exhibited not only high loading but also controlled release behavior. The cytotoxicity of the GPNPs was evaluated by incubating with HEK293T cell lines and they were shown to be of low cytotoxicity. They appear to be a very promising candidate for targeted drug delivery.

Hypoxia-inducible factor-1 is a positive regulator of Sox9 activity in femoral head osteonecrosis.

Legg-Calve-Perthes disease (LCPD) is a juvenile form of ischemic osteonecrosis of the femoral head leading to femoral head deformity and premature osteoarthritis. Femoral head osteonecrosis occurs due to blood supply disruption which results in hypoxic injury to the femoral head. Hypoxia-inducible factor-1 α (HIF-1α) is a master regulator of cellular response to hypoxia. A pig model of ischemic osteonecrosis of femoral head has been shown to have radiographic and histopathologic changes resembling LCPD. Our preliminary studies showed that the cartilage layer was thicker in the hypoxia group compared to the control group. The mechanism underlying this cartilage response is not known. To explore the hypoxia-induced downstream gene activity following the femoral head ischemia, porcine microarray analysis of gene profiles of chondrocytes from normal and ischemic femoral heads was performed. In the ischemic side, the expression of Sox9, a transcription factor required for chondrocyte differentiation, was upregulated along with HIF-1α. Expressions of Sox9 target genes, such as type II collagen and aggrecan, were also increased. Microarray results were confirmed by quantitative real-time RT-PCR. In addition, immunohistochemistry assay demonstrated that both HIF-1α and Sox9 were upregulated in chondrocytes in ischemic femoral heads compared with normal controls. To investigate the possible molecular mechanisms of hypoxia on Sox9 activity, we tested the effect of HIF-1α on Sox9 expression in vitro. We made a luciferase reporter construct driven by 2kb Sox9 promoter. Transient transfection assay showed that HIF-1α activated Sox9 promoter activity in a dose-dependent manner. Sox9 is known to activate type II collagen target gene expression. To test the effect of HIF-1α on Sox9-mediated transcription, HIF-1α was cotransfected with Sox9 in type II collagen reporter assay. Our results demonstrated that HIF-1α enhanced Sox9-mediated transcriptional activity. Moreover, coimmunoprecipitation assay showed that HIF-1α associated with Sox9 directly. Taken together, these findings indicate that HIF-1α activates Sox9 expression and enhances Sox9-mediated transcriptional activity and that HIF-1α physically interacts with Sox9. We speculate that HIF-1α upregulation of Sox9 activity may have a chondroprotective role following femoral head ischemia.

Androgen receptor-mediated regulation of the anti-atherogenic enzyme CYP27A1 involves the JNK/c-jun pathway.

CYP27A1, an enzyme with several important roles in cholesterol homeostasis and vitamin D3 metabolism, has been ascribed anti-atherogenic properties. This study addresses an important problem regarding how this enzyme, involved in cholesterol metabolism in the liver and peripheral tissues, is regulated. Our results identify the human CYP27A1 gene as a new target for the JNK/c-jun pathway. Initial experiments showed that an inhibitor of c-Jun N-terminal kinase (JNK) downregulated basal CYP27A1 promoter activity whereas overexpression of JNK slightly enhanced promoter activity. Androgen receptor (AR)-mediated upregulation of mRNA levels and endogenous enzyme activity was recently reported. In the present study, the AR antagonist nilutamide blocked the androgen induction of CYP27A1. The present data revealed that inhibition of the JNK/c-jun pathway abolishes the AR-mediated effect on CYP27A1 transcription and enzyme activity, whereas overexpression of JNK markedly increased androgenic upregulation of CYP27A1. In conclusion, the current results indicate involvement of the JNK/c-jun pathway in AR-mediated upregulation of human CYP27A1. The link to JNK signaling is interesting since inflammatory processes may upregulate CYP27A1 to clear cholesterol from peripheral tissues.

Sclerostin is a direct target of osteoblast-specific transcription factor osterix.

Osterix (Osx) is an osteoblast-specific transcription factor required for osteoblast differentiation and bone formation. Osx knock-out mice lack bone completely. Recent findings that Osx inhibits Wnt signaling provide a feedback control mechanism involved in bone formation. Mechanisms of Osx inhibition on Wnt signaling are not fully understood. Our results in this study revealed that the expression of a Wnt antagonist Sclerostin (Sost) was downregulated in Osx-null calvaria. Overexpression of Osx in stable C2C12 mesenchymal cell line resulted in Sost upregulation. Transient transfection assay showed that Osx activated 1kb Sost promoter reporter activity in a dose-dependent manner. To define Sost promoter activated by Osx, we made a series of deletion mutants of Sost constructs, and narrowed down the minimal region to the proximal 260bp. Gel shift assay indicated that Osx bound to GC-rich site within this minimal region, and that point mutations of this binding site disrupted Osx binding. Moreover, the same point mutations in 260bp Sost promoter reporter disrupted the promoter activation by Osx, suggesting that the GC-rich binding site was responsible for Sost promoter activation by Osx. To further examine physical association of Osx with Sost promoter in vivo, Chromatin immunoprecipitation (ChIP) assays were performed using primary osteoblasts from mouse calvaria. Osx was found to associate with endogenous Sost promoter. Taken together, these findings support our hypothesis that Sost is a direct target of Osx. This provides a new additional mechanism through which Osx inhibits Wnt signaling during bone formation.

Involvement of the PI3K/Akt pathway in estrogen-mediated regulation of human CYP7B1: identification of CYP7B1 as a novel target for PI3K/Akt and MAPK signalling.

The steroid hydroxylase CYP7B1 metabolizes neurosteroids, cholesterol derivatives, and estrogen receptor (ER) ligands. Previous studies identified CYP7B1 as a target for regulation by estrogen. The present study examines the mechanism for estrogen-mediated regulation of the human CYP7B1 gene promoter. Treatment with LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), abolished ER-mediated up-regulation of a CYP7B1 promoter-luciferase reporter in HepG2 cells, whereas overexpression of PI3K or Akt significantly increased estrogenic up-regulation of CYP7B1. Overexpression of dominant-negative mutant Akt abolished ER-mediated stimulation of CYP7B1 in HepG2 cells. Data indicated no binding of ER to CYP7B1 promoter sequences, suggesting that ER interacts with the PI3K/Akt pathway without binding to the gene. At low ER levels, overexpression of Akt suppressed CYP7B1 promoter activity, suggesting that its effect on CYP7B1 is different when estrogens are absent. In HEK293 cells, CYP7B1 transcription was much less affected by Akt, indicating that the mechanism for up-regulation of CYP7B1 is different in different cell types. Other experiments indicated that MAPK signalling may affect basal CYP7B1 levels. The current results, indicating that regulation of CYP7B1 by ER can be mediated via the PI3K/Akt signal pathway, a regulatory pathway important for cellular survival and growth, suggest an important role for CYP7B1 in cellular growth, particularly in connection with estrogenic signalling.