Do biomarkers allow a differentiation between osteonecrosis of the femoral head and osteoarthritis of the hip? - a biochemical, histological and gene expression analysis.

OBJECTIVE: Osteonecrosis of the femoral head (ONFH) is a devastating disease of the hip joint. Its early diagnosis is crucial to increase the chances of joint preserving, yet difficult due to similarities with osteoarthritis (OA) of the hip in its clinical appearance. The purpose of this study was to enhance the understanding of ONFH and its pathologic processes in contrast to OA and to identify serum biomarkers helping to improve the diagnosis of the disease. DESIGN: Bone and bone marrow samples were collected from 24 patients diagnosed with OA and 25 patients with ONFH during total hip replacement surgery. RNA was isolated, histological examination, determination of free reactive oxygen species as well as gene expression and biomarker analysis were performed. RESULTS: Histological analysis revealed differences in the structural and cellular pattern between the groups. Gene expression analysis revealed a significant upregulation for the genes ASPN, COL1A1, COL2A1 and IL6 and a significant downregulation for HIF1A in ONFH compared to OA group. Analysis of serum biomarkers showed significant differences between the groups for asporin and adiponectin. A final logistical regression model including the parameters adiponectin, asporin and HIF 1α was overall significant, explained 34.5 % of variance and classified 74.5 % of the cases correctly. CONCLUSION: The combination of adiponectin, asporin and HIF 1α as serum biomarkers revealed a classification accuracy of 74.5 %. The information provided in this study may help to enhance the understanding of pathologic processes in ONFH and to elaborate further aspects of prediction and treatment.

Autoregulation enables different pathways to control CCAAT/enhancer binding protein beta (C/EBP beta) transcription.

CCAAT/enhancer binding protein beta (C/EBP beta) also named liver-enriched transcriptional activating protein (LAP) is a member of the C/EBP family of transcription factors and is involved in hepatocyte-specific gene expression and in the process of tissue differentiation. The activity of LAP/C/EBP beta can be regulated at the transcriptional and posttranslational level or by protein-protein interaction with other transcription factors. In this study we show that LAP/C/EBP beta can stimulate its own transcription. Deletion analysis of the rat LAP/C/EBP beta promoter in luciferase reporter gene experiments demonstrated that the region located between nucleotide -121 to -71, comprising two recently characterized cAMP responsive element (CRE)-like elements, is important for autoregulation. Gel shift experiments using oligonucleotides with overlapping point mutations identified the sequence GCAATGA (beta-site) adjacent to and partially overlapping the first CRE-like site as core motif for LAP/C/EBP beta binding. Analysis of a mutated beta-site in reporter gene experiments showed the functional relevance of this site for autoregulation. The composite C/EBP beta-CRE-element in the promoter enables synergistic activation of transcription by LAP/C/EBP beta and the protein kinase A (PKA)/cAMP responsive element binding protein (CREB) pathway in a cell-type specific manner. In hepatoma cells nuclear factor kappa B (NF-kappa B) increased autoregulation and therefore could mediate enhanced activation during inflammatory responses. In summary, our results demonstrated that the assembly of the three binding sites in the promoter and thus the interaction between LAP/C/EBP beta and members of the CREB or NF-kappa B family allows the control of LAP/C/EBP beta gene transcription as a response to different stimuli in a tissue specific manner.

Interleukin-6-induced tethering of STAT3 to the LAP/C/EBPbeta promoter suggests a new mechanism of transcriptional regulation by STAT3.

LAP/C/EBPbeta is a member of the C/EBP family of transcription factors and contributes to the regulation of the acute phase response in hepatocytes. Here we show that IL-6 controls LAP/C/EBPbeta gene transcription and identify an IL-6 responsive element in the LAP/C/EBPbeta promoter, which contains no STAT3 DNA binding motif. However, luciferase reporter gene assays showed that STAT3 activation through the gp130 signal transducer molecule is involved in mediating IL-6-dependent LAP/C/EBPbeta transcription. Southwestern analysis indicated that IL-6 induces binding of a 68-kDa protein to the recently characterized CRE-like elements in the LAP/C/EBPbeta promoter. Transfection experiments using promoter constructs with mutated CRE-like elements revealed that these sites confer IL-6 responsiveness. Further analysis using STAT1/STAT3 chimeras identified specific domains of the protein that are required for the IL-6-dependent increase in LAP/C/EBPbeta gene transcription. Overexpression of the amino-terminal domain of STAT3 blocked the IL-6-mediated response, suggesting that the STAT3 amino terminus has an important function in IL-6-mediated transcription of the LAP/C/EBPbeta gene. These data lead to a model of how tethering STAT3 to a DNA-bound complex contributes to IL-6-dependent LAP/C/EBPbeta gene transcription. Our analysis describes a new mechanism by which STAT3 controls gene transcription and which has direct implication for the acute phase response in liver cells.

The designer cytokine hyper-interleukin-6 is a potent activator of STAT3-dependent gene transcription in vivo and in vitro.

Interleukin-6 (IL-6) triggers pivotal pathways in vivo. The designer protein hyper-IL-6 (H-IL-6) fuses the soluble IL-6 receptor (sIL-6R) through an intermediate linker with IL-6. The intracellular pathways that are triggered by H-IL-6 are not defined yet. Therefore, we studied the molecular mechanisms leading to H-IL-6-dependent gene activation. H-IL-6 stimulates haptoglobin mRNA expression in HepG2 cells, which is transcriptionally mediated as assessed by run-off experiments. The increase in haptoglobin gene transcription correlates with higher nuclear translocation of tyrosine-phosphorylated STAT3 and its DNA binding. As H-IL-6 stimulates STAT3-dependent gene transcription, we compared the molecular mechanism between IL-6 and H-IL-6. Transfection experiments were performed with a STAT3-dependent luciferase construct. The same amount of H-IL-6 stimulated luciferase activity faster, stronger, and for a longer period of time. Dose response experiments showed that a 10-fold lower dose of H-IL-6 stimulated STAT3-dependent gene transcription comparable with the higher amount of IL-6. Cotransfection with the gp80 and/or gp130 receptor revealed that the effect of H-IL-6 on STAT3-dependent gene transcription is restricted to the gp80/gp130 receptor ratio. High amounts of gp130 increased and high amounts of gp80 decreased the effect on H-IL-6-dependent gene transcription. To investigate the in vivo effect of H-IL-6 on gene transcription in the liver, H-IL-6 and IL-6 were injected into C3H mice. H-IL-6 was at least 10-fold more effective in stimulating the DNA binding and nuclear translocation of STAT3, which enhances haptoglobin mRNA and protein expression. Thus H-IL-6 stimulates STAT3-dependent gene transcription in liver cells in vitro and in vivo at least 10-fold more effectively than IL-6. Our results provide evidence that H-IL-6 is a promising designer protein for therapeutic intervention during different pathophysiological conditions also in humans.

CREB controls LAP/C/EBP beta transcription.

LAP/C/EBP beta is a member of the C/EBP family of transcription factors and is involved in hepatocyte-specific gene expression. Recently we showed that, besides its posttranscriptional regulation, LAP/C/EBP beta mRNA is modulated during liver regeneration. Therefore, in this study we investigated mechanisms which control LAP/C/EBP beta gene transcription. Deletion analysis of the 5'-flanking region, located upstream of the start site of transcription in the LAP/C/EBP beta gene, demonstrated that a small region in close proximity to the TATA box is important in maintaining a high level of transcription of the luciferase reporter gene constructs. In gel shift experiments two sites were identified which are important for specific complex formation within this region. Further analysis by cross-linking, super shift, and competition experiments was performed with liver cell nuclear extracts, hepatoma cell nuclear extracts, or recombinant CREB protein. These experiments conclusively demonstrated that CREB binds to both sites in the LAP/C/EBP beta promoter with an affinity similar to that with the CREB consensus sequence. Transfection experiments with promoter constructs where the CREB sites were mutated showed that these sites are important to maintain both basal promoter activity and LAP/C/EBP beta inducibility through CREB. Northern blot analysis and runoff transcription assays demonstrated that the protein kinase A pathway not only stimulated the activity of the luciferase reporter construct but also the transcription of the endogenous LAP/C/EBP beta gene in different cell types. Western blot analysis of rat liver cell nuclear extracts and runoff transcription assays of rat liver cell nuclei after two-thirds hepatectomy showed a functional link between the induction of CREB phosphorylation and LAP/C/EBP beta mRNA transcription during liver regeneration. These results demonstrate that the two CREB sites are important to control LAP/C/EBP beta transcription in vivo. As several pathways control CREB phosphorylation, our results provide evidence for the transcriptional regulation of LAP/C/EBP beta via CREB under different physiological conditions.

Target gene modulation in hepatocellular carcinomas by decreased DNA-binding of p53 mutations.

The crystallographic structure of the p53 core domain showed that most of the p53 mutations found in human tumors are located in conserved regions of the p53 DNA-binding domain. The aim of our study was to investigate the effect on DNA-binding and transactivation of three p53 mutations frequently found in hepatocellular carcinomas (HCC). Two of these mutations are located near the DNA-binding surface and are induced by aflatoxin B1 (249ser) and oxiradicals (249met). In contrast, mutation 220cys is not associated with a specific carcinogen in HCCs and is located outside the DNA binding structures of p53. Cotransfection experiments in two HCC cell lines, with mutated or deleted P53 genes, showed that all three mutations did not enhance reporter gene activity (RGC-CAT), in contrast to wt p53. However, in hepatoma cell lines all three mutations did suppress the p53 wildtype (wt) transactivation in a dose-dependent fashion. DNA-binding was monitored by gel shift assays using the consensus-, Waf-, and RGC-p53 binding sites. All three p53 mutations did decrease DNA-binding versus all binding sites included. Interestingly although all mutations showed the same DNA-binding and transactivation properties, differences in the ectopic expression in different hepatoma cells were observed. Therefore our results indicate that p53 mutations in HCC found in the DNA-binding domain and outside the conserved DNA-binding structures modulate target gene expression by decreasing sequence specific DNA-binding in a dominant negative fashion. The cellular environment may contribute to an additional selection advantage of some mutations.

Differences in the regulation of cytochrome P450 family members during liver regeneration.

BACKGROUND/AIMS: Cytochrome P450 enzymes (P450s) metabolise endogenous substances and a vast variety of drugs. Little is known about the regulation of P450s during pathophysiological conditions in the liver. Therefore we studied the regulation of P450 1A1, 1A2, 2E1 and 3A during liver regeneration after two-thirds hepatectomy. METHODS: Partial hepatectomy or sham surgery was performed in Sprague-Dawley rats. At different time points after surgery, microsomal proteins were isolated and the RNA was prepared. Northern blot analysis, Western blot analysis and enzyme assays for the different P450s were performed. RESULTS: Northern blot analysis showed a transient downregulation of cytochromes P450 1A2 and 2E1 after hepatectomy, while the expression of cytochrome P450 3A remained unaffected. Western blot analysis of microsomal proteins showed that changes of the mRNA levels are not reflected in the protein level, most likely because the half-life of the P450 proteins in hepatocytes is long, and thus a transient mRNA downregulation has little impact on the total amount of protein detected. Differences in the regulation of the enzymatic activities were found for P450 1A2 and 2E1. Interestingly, the metabolic activity of cytochrome P450 2E1 decreased dramatically post-hepatectomy, while the P450 2A1 activity remained unchanged. CONCLUSIONS: Regulatory mechanisms were found on the RNA level and by post-translational mechanisms which downregulate P450 expression and activity during liver regeneration. These results indicate prolonged half-life of drugs during hepatocyte proliferation, and thus also have important implications for therapy in humans.

Acute-phase response factor, increased binding, and target gene transcription during liver regeneration.

BACKGROUND & AIMS: The acute-phase response may contribute and influence cell-cycle progression in hepatocytes. The aim of this study was to examine the regulation of the alpha 2-macroglobulin gene during liver regeneration and molecular mechanisms that influence its expression. METHODS: Partial hepatectomy or sham surgery was performed in Sprague-Dawley rats. At different time points after surgery blood was taken from the liver vein, and nuclear extracts and RNA were prepared. Northern blot analysis, run-off assays, gel shift experiments, and cytokine assays were performed. RESULTS: Increased transcription of the alpha 2-macroglobulin gene was found 12-24 hours posthepatectomy and not after sham surgery. Increased levels of alpha 2-macroglobulin messenger RNA correlated with enhanced binding of acute-phase response factor/signal transducer and activator of transcription 3 (APRF/Stat3) towards the cognate DNA sequence in the alpha 2-macroglobulin promoter and dramatically increased interleukin-6 levels in the liver vein. In contrast, nuclear translocation of APRF/Stat3 was detected as early as 1 hour after hepatectomy and up to 48 hours posthepatectomy. Therefore, two events can be distinguished in the regulation of APRF/Stat3: Its nuclear translocation and increased DNA binding. CONCLUSIONS: Increased alpha 2-macroglobulin transcription posthepatectomy is achieved by increased levels of interleukin 6 and consecutive binding of APRF/Stat3 to the alpha 2-macroglobulin promoter. A two-step event is suggested for APRF/Stat3-dependent gene activation in hepatocytes.

Direct interaction and N-terminal phosphorylation of c-Jun by c-Mil/Raf.

c-Mil is the avian homologue of the mammalian serine/threonine kinase c-Raf-1. c-Mil/Raf is a mediator of signal transduction leading to gene expression via the c-Jun DNA-binding site, AP-1. Here we show that c-Mil immunopurified from MC29-virus-transformed quail fibroblasts phosphorylates c-Jun in vitro near its N terminus (Ser-63 and -73). Furthermore, the viral oncogene product Gag-Mil of the avian wild-type retrovirus MH2 phosphorylates c-Jun in vitro. A contribution by other known kinases phosphorylating c-Jun, such as the mitogen-activated protein kinases (MAPKs) and the c-Jun N-terminal kinases, was excluded by control reactions. c-Raf-1 and c-Jun directly interact in vitro as shown by various immobilized glutathione S-transferase-Raf fusion proteins which specify the cysteine-rich region of c-Mil/Raf as the major N-terminal binding site. An additional minor binding site is located in the C-terminal region. The biological relevance of these results is demonstrated by coimmunoprecipitation of c-Jun and c-Mil from 32P-labeled MC29- and MH2-transformed fibroblasts as well as normal quail embryo fibroblasts, whereby c-Jun was identified by tryptic phosphopeptide analysis. The complexed c-Jun exhibits a decreased electrophoretic mobility corresponding to a more highly phosphorylated state. Cell fractionation analyses indicate that the c-Mil/c-Jun complex is located in the cytoplasm. The data demonstrate that c-Jun can be a direct target of the protein kinase c-Mil/Raf, suggesting an alternative pathway, which leads to c-Jun phosphorylation independent of the MAPKs and MAPK-related proteins.

A small peptide derived from the aminoterminus of c-Raf-1 inhibits c-Raf-1/Ras binding.

Various domains of the aminoterminal part of c-Raf-1 expressed as glutathione-S-transferase fusion proteins were analyzed for Ras binding. The binding site was localized at the aminoterminus outside of the cysteine-rich region. A single aminoacid exchange at aminoacid residue 89 (Arg89 to Leu) of c-Raf-1 inhibits binding. A small synthetic peptide corresponding to c-Raf-1 aminoacids 77 to 101 comprising Arg89 in a central position competes for Ras binding and thereby characterizes the relevant binding domain of Ras on c-Raf-1.

The aminoterminus of c-Raf-1 binds a protein kinase phosphorylating Ser259.

The kinase negative aminoterminal domain of c-Raf-1 expressed as glutathione S-transferase fusion protein was phosphorylated in vitro after treatment with lysates from A431 cells and subsequent in vitro protein kinase assay. This phosphorylation was independent of stimulation of the cells with EGF; it occurred exclusively on serine and was mapped to Ser259. The identical site of c-Raf-1 was phosphorylated in A431 cells by metabolic labelling in vivo. The kinase binding domain was mapped by various GST-Raf deletion mutants to c-Raf-1 aminoacid residues 181 to 255.

The aminoterminus of c-Raf-1 binds a 28-kD cellular protein.

The regulatory aminoterminal domain of c-Raf-1 expressed as glutathione S-transferase fusion protein associates with a 28-kD cellular protein after treatment with lysates from A431 cells. Both proteins become phosphorylated in vitro by an unidentified cellular protein kinase also present in the complex. The association of the 28-kD protein depends on Cys 168 and Ser 259, suggesting that two independent epitopes of c-Raf-1 are required for binding.

Phosphorylation of c-Raf-1 by protein kinase A interferes with activation.

c-Raf-1 is a serine/threonine-specific protein kinase which is regulated by phosphorylation. A putative c-AMP dependent protein kinase PKA phosphorylation site with the consensus sequence RRXS, Ser43, and a predominant phosphorylation site of c-Raf-1, Ser259, can be phosphorylated by PKA in vitro as shown by comparison of phosphopeptide maps of recombinant wild-type c-Raf-1 and the corresponding mutants. In vivo stimulation of the PKA pathway by treatment of A431 cells with Forskolin results in increase of phosphorylation in Ser43. Forskolin reduces the upshift of c-Raf-1 induced by EGF-treatment. It inhibits the EGF-activation of the c-Raf-1 protein kinase activity tested in vitro with a peptide substrate.