Stress-specific responses of p21 expression: implication of transcript variant p21 alt-a in long-term hypoxia.

p21 (CDKN1A, Cip1, Waf1) is a cyclin-dependent kinase inhibitor capable of causing cell cycle arrest or promoting cell cycle transit as well as acting as a regulator of apoptosis. In this study, we analyzed the effects of various antemortem conditions on p21 protein level and expression profiles of known p21 transcript variants in human heart tissue. The selected death cause groups were: non-cardiac, hypothermia, acute ischemia, and chronic hypoxia. Immunohistochemical staining of p21 in cardiac myocytes could be observed only in hypothermia death cases, in which the mRNA expression of the most abundant variant, p21V1, also exceeded that in other death cause groups. Cytoplasmic localization of p21 protein in vascular smooth muscle cells together with substantially increased expression of cardioprotective Pim-1 especially in chronic hypoxia, but in acute ischemia and hypothermia as well, indicate change of p21 function from cell cycle arrest to promotion of proliferation and cell survival in these cases. In chronic hypoxia deaths the expression of variant p21 alt-a was highly pronounced whereas the expression of variant p21B was low. In chronic hypoxia deaths the expression of p53 was substantially higher compared to the other groups, being a potential regulator of p21 alt-a expression. In acute ischemia deaths increased expression of variant p21B, suggested to be proapoptotic in several cell lines, was observed. Our results suggest a role for variant p21 alt-a in hypoxia and for variant p21B in acute myocardial ischemia. The known cardioprotective aspect of hypothermia might come from an increased p21 protein level.

Adiponectin induced placental cell apoptosis could be mediated via the ADIPOR1-receptor in pre-eclampsia with IUGR.

AIMS: Adiponectin and leptin are members of the adipocytokine family. Adiponectin promotes and leptin inhibits apoptosis and both are regulators of angiogenesis. Adipocytokines and their receptors are expressed in the placenta, and in the pre-eclamptic (PE) mother the serum levels of both are higher than in healthy ones. Our aim was to study the expression of adiponectin, leptin, their receptor genes and apoptosis in severely PE and normal placentas. METHODS: The study group comprised 13 PE mothers and their 16 healthy controls. Placental biopsies were taken during cesarean section, the RNA was extracted and micro-array study was performed, followed by PCR and apoptosis studies. RESULTS: The placental expression level of the leptin and adiponectin receptor 1 genes was significantly higher in PE mothers than in controls. No significant changes were observed in the levels of the adiponectin, adiponectin receptor 2 and Leptin receptor genes. The expression of the Adiponectin gene was low. The rate of apoptosis was higher in the PE placentas. CONCLUSIONS: The activity of placental adipocytokines and their receptor genes in severe PE may suggest an important role in placental angiogenesis. Placental apoptosis induced by adiponectin could be mediated via the ADIPOR1-receptor.

General expression profiles of human native odontoblasts and pulp-derived cultured odontoblast-like cells are similar but reveal differential neuropeptide expression levels.

OBJECTIVES: Odontoblasts play a central role during the dentin formation by organic matrix production and mineralisation. Recently, suitable in vitro techniques for studying mature primary odontoblasts and the newly differentiated odontoblasts have been developed. Firstly, the gene expression profiles of native and cultured odontoblasts were compared at large-scale to investigate the similarities and differences between the samples. Secondly, differential expression levels of the genes encoding neuronal proteins were analyzed to study odontoblasts sensory function. DESIGN: Microarray analysis was performed to mature native and cultured pulp-derived odontoblast-like cells to compare their transcriptome. Then, the probes positive only in one sample were divided into gene ontology categories. Expression levels of selected neuronal proteins were further studied with quantitative PCR, and at the protein level by immunofluorescence of mature and newly differentiated odontoblasts in developing tooth. RESULTS: Remarkable similarities between the general and neuronal protein gene expression profiles were observed. Higher cortistatin, galanin, somatostatin receptor 1 (SSTR1) and tyrosine phosphatase receptor type Z1 (PTPRZ1) expression was detected in native than in cultured odontoblast at the mRNA level. Pronociceptin was more abundantly expressed in cultured than in native odontoblasts. Immunofluorescence of mature and newly differentiated odontoblasts on human tooth germs confirmed the results. CONCLUSIONS: Cultured odontoblasts used in this study have similar general gene expression pattern to native odontoblasts, and therefore offer a valuable tool for the in vitro odontoblast studies. The expression of PTPRZ1 and galanin, which participate in sensory signal transduction, supports the previously suggested role of odontoblasts as sensory cells.

Immunological detection of in vitro formed phosphatidylethanol--an alcohol biomarker--with monoclonal antibodies.

BACKGROUND: Phosphatidylethanol (PEth) is a promising new marker for detecting long-term alcohol abuse with excellent sensitivity and specificity. Current methods are based on the high performance liquid chromatography-mass spectrometric method and therefore require high levels of expertise and expensive instrumentation. This study was designed to generate PEth-specific monoclonal antibodies for PEth immunoassay development. METHODS: C57/BL6 mice were immunized with PEth in 3 different carriers, mouse serum albumin, mouse high-density lipoproteins, and human low-density lipoprotein (LDL). Mouse splenocytes were fused with a mouse myeloma cell line using the hybridoma technique. Mouse IgM-producing cell lines were selected by limiting dilutions. Binding characteristics of the anti-PEth antibodies were studied using luminometric immunoassays and sequence analysis of the variable region mRNA sequences of the antibodies. Produced antibodies were purified by chromatographic methods. PEth was detected with these antibodies in fluorescence immunoassay and flow cytometric analysis. RESULTS: We generated monoclonal cell lines (2B1 and 2E9) that produce IgM antibodies binding specifically to PEth but not to structurally or chemically similar phospholipids, such as phosphatidylcholine, phosphatidic acid, and cardiolipin. We show here that these anti-PEth antibodies can be used to detect PEth in a fluorescent PEth assay and FACS analysis of human red blood cell samples spiked with PEth. CONCLUSIONS: The present study shows that PEth-specific monoclonal antibodies can be generated using traditional hybridoma technique. Immunogenicity of PEth was enhanced using human LDL as an immunization carrier. The generated monoclonal anti-PEth antibodies, 2B1 and 2E9 bind to PEth in fluid phase and in biological membranes.

Altered expression of angiogenesis-related placental genes in pre-eclampsia associated with intrauterine growth restriction.

AIM: The normal endovascular invasion of trophoblast cells and spiral artery remodeling are impaired in pre-eclampsia. Neither the circulating factor secreted by the placenta nor the cause of the widespread endothelial dysfunction in pre-eclampsia has yet been identified. In an attempt to identify novel factors, we performed a gene expression profiling study of placental tissue from women with and without pre-eclampsia. MATERIAL AND METHODS: The study group comprised two pre-eclamptic patients with intrauterine growth restriction while the control group comprised three healthy women with uncomplicated pregnancies. Gene expression was studied using Affymetrix Human Genome U133 Plus 2 micro arrays. We focused on genes associated with angiogenesis. Some of the micro array analysis results were verified using real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Gene expression profiling revealed that the expression level of nine genes--ECGF1, JAG1, Palladin, COL18A1, TNFSF12, VEGF, ANPEP, PDGFRA and SERPIN12 - was downregulated whereas the level of four genes--EPAS1, FLT1, SIGLE10 and ANG4--was upregulated in the study group compared with the control group. The real-time RT-PCR results from JAG1, COL18A1 and FLT1 genes were in accordance with the gene expression results. CONCLUSION: Our results show new targets for research to understand the mechanisms leading to pre-eclampsia.

Effects of phosphatidylethanol on mouse adipocyte differentiation and expression of stearoyl-CoA desaturase 1.

BACKGROUND: Phosphatidylethanol (PEth) is an aberrant phospholipid formed in vivo only in the presence of ethanol. In circulation PEth is associated with lipoproteins and is transferred from one lipoprotein to another. Lipoprotein-associated PEth affects endothelial and smooth muscle cells of blood vessels, but its effects on other cell types have not been explored. Adipocytes have a central role in metabolic syndrome and obesity. In this study we tested whether lipoprotein-associated PEth affects stearoyl-CoA desaturase 1 (SCD1) which plays a major role in lipid-mediated signaling in the differentiation of adipocytes. METHODS: Mouse 3T3-L1 preadipocytes were differentiated to adipocytes in the presence of high-density lipoproteins (HDL) isolated from the plasma of healthy volunteers or PEth-containing HDL modified in vitro. After incubation, fat accumulation, SCD1 mRNA expression, SCD1 protein content, and fatty acid composition of adipocytes were determined. RESULTS: Phosphatidylethanol-containing HDL particles inhibited adipocyte differentiation and decreased the 18:1/18:0 ratio of cellular fatty acids by 28% compared with native HDL particles. Moreover, PEth-containing HDL reduced the SCD1 protein content by 39%. CONCLUSIONS: Lipoprotein-associated PEth may mediate the effects of ethanol on SCD1 and differentiation of preadipocytes to adipocytes.

Characterization of androgen-regulated expression of CYP3A5 in human prostate.

Testosterone is needed for the growth and development of the prostate. Androgen deprivation therapy is used for the treatment of prostate cancer. CYP3A5 is a human drug-metabolizing cytochrome P450 enzyme that metabolizes testosterone to the inactive 6beta-hydroxylated metabolite. We identified CYP3A5 as a novel androgen-regulated gene in human prostate by GeneChip analysis of human prostate tissues obtained from patients 3 days after therapeutic castration and from control patients. We further showed androgen induction of CYP3A5 messenger RNA (mRNA) in LNCaP prostate cancer cell line. Immunoblotting studies revealed CYP3A5 protein expression in all prostate samples studied. Immunohistochemistry and in situ hybridization was used for localization of CYP3A5 expression in prostate tissue. CYP3A5 was detected both in luminal and in basal epithelial cells of human prostate. Androgen response element was identified in the CYP3A5 proximal promoter and in electrophoretic mobility shift assay androgen receptor was found to bind this element. Androgen induction was abolished by mutation of the response element. We suggest that CYP3A5 is a part of an autoregulatory feedback loop controlling prostate cell exposure to androgens.

Use of differentiating adult stem cells (marrow stromal cells) to identify new downstream target genes for transcription factors.

We developed a strategy for use of microarray data to rapidly identify new downstream targets of transcription factors known to drive differentiation by following the time courses of gene expression as a relatively homogeneous population of stem/progenitor cells are differentiated to multiple phenotypes. Microarray assays were used to follow the differentiation of human marrow stromal cells (MSCs) into chondrocytes or adipocytes in three different experimental conditions. The steps of the analysis were the following: (a) hierarchical clustering was used to define groups of similarly behaving genes in each experiment, (b) candidates for new downstream targets of transcription factors that drive differentiation were then identified as genes that were consistently co-expressed with known downstream target genes of the transcription factors, and (c) the list of candidate new target genes was refined by identifying genes whose signal intensities showed a highly significant linear regression with the signal intensities of the known targets in all the data sets. Analysis of the data identified multiple new candidates for downstream targets for SOX9, SOX5, CCAAT/enhancer binding protein (C/EBP)-alpha, and peroxisome proliferator-activated receptor (PPAR)-gamma. To validate the analysis, we demonstrated that PPAR-gamma protein specifically bound to the promoters of four new targets identified in the analyses. The same multistep analysis can be used to identify new downstream targets of transcription factors in other systems. Also, the same analysis should make it possible to use MSCs from bone marrow to define new mutations that alter chondogenesis or adipogenesis in patients with a variety of syndromes.

Comparison of effect of BMP-2, -4, and -6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma.

The human adult stem cells from bone marrow stroma referred to as mesenchymal stem cells or marrow stromal cells (MSCs) are of interest because they are easily isolated and expanded and are capable of multipotential differentiation. Here, we examined the ability of recombinant human bone morphogenetic protein (BMP)-2, -4, and -6 to enhance in vitro cartilage formation of MSCs. Human MSCs were isolated from bone marrow taken from normal adult donors. The cells were pelleted and cultured for 21 days in chondrogenic medium containing transforming growth factor beta3 and dexamethasone with or without BMP-2, -4, or -6. All the BMPs tested increased chondrogenic differentiation as assayed by immunohistochemistry and by the size and weight of the cartilage synthesized. However, BMP-2 was the most effective. Microarray analyses of approximately 12,000 genes and reverse transcription-polymerase chain reaction assays established that the critical genes for cartilage synthesis were expressed in the expected time sequence in response to BMP-2. The tissue engineering of autologous cartilage derived from MSCs in vitro for transplantation will be a future alternative for patients with cartilage injuries. To obtain large amounts of cartilage rich in proteoglycans, the use of BMP-2 is recommended, instead of BMP-4 or -6.

Adipogenic differentiation of human adult stem cells from bone marrow stroma (MSCs).

UNLABELLED: We assayed gene expressions during adipogenesis of human MSCs. Microarray assays demonstrated time-dependent increases in expression of 67 genes, including 2 genes for transcription factors that were not previously shown to be expressed during adipogenesis. INTRODUCTION Increased numbers of bone marrow adipocytes have been observed in patients with osteoporosis and aplastic anemia, but the pathological mechanisms remain unknown. Recently, microarray assays for mRNAs were used to follow adipogenic differentiation of the preadipocytic cell line, 3T3-L1, but adipogenic differentiation has not been examined in primary cells from bone marrow. Here we defined the sequence of gene expression during the adipogenesis ex vivo of human cells from bone marrow referred to as either mesenchymal stem cells or marrow stromal cells (MSCs). MATERIALS AND METHODS: MSCs were plated at extremely low densities to generate single-cell derived colonies, and adipogenic differentiation of the colonies assayed by accumulation of fat vacuoles, time-lapse photomicroscopy, microarrays, and reverse transcriptase-polymerase chain reaction (RT-PCR) assays. RESULTS AND CONCLUSIONS About 30% of the colonies differentiated to adipocytes in 14 days and about 60% in 21 days. Cell proliferation was inhibited by approximately 50% in adipogenic medium. The differentiation occurred primarily at the center of the colonies, and a few adipocytes that formed near the periphery migrated toward the centers. RT-PCR assays demonstrated that the differentiation was accompanied by increases in a series of genes previously shown to increase with adipogenic differentiation: peroxisome proliferator activated receptor gamma, CCAAT enhancer-binding protein alpha, acylCoA synthetase, lipoprotein lipase, and fatty acid binding protein 4. We also followed differentiation with microarray assays. Sixty-seven genes increased more than 10-fold at day 1 and 20-fold at day 7, 14, or 21. Many of the genes identified were previously shown to be expressed during adipocytic differentiation. However, others, such as zinc finger E-box binding protein and zinc finger protein 145, were not. This study should serve as a basis for future study to clarify the mechanisms of adipocyte differentiation of MSCs.

In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis.

One approach to resolving the complexities of chondrogenesis is to examine simplified systems in vitro. We analyzed cartilage differentiation by human adult stem cells from bone marrow stroma. Marrow stromal cells were cultured as micromass pellets for 21 days in serum-free medium containing transforming growth factor (TGF)-beta3, dexamethasone, and bone morphogenetic protein (BMP)-6. Assays for pulse-labeled [3H]DNA and for total DNA indicated that there was little proliferation and a progressive loss of cells in the pellets. There were continuous increases in mRNAs for cartilage matrix (proteoglycans and COL2, -9, -10, and -11), receptors [fibroblast growth factor 2 (FGFR2) and parathyroid hormone-related peptide receptor (PTHrP-R)], and transcription factors (SOX5, -6, and -9) as demonstrated by histochemical and microarray assays. Reverse transcription-PCR assays for 11 mRNAs confirmed the microarray data. SOX4, vascular endothelial growth factor (VEGF), and matrix metalloproteinase 14 (MMP14) increased at day 1 and decreased thereafter, suggesting roles early in chondrogenesis. Also, forkhead, CD10, and MMP13 increased up to day 7 and decreased thereafter, suggesting roles in an intermediate stage of chondrogenesis. In addition, two collagens (COL3A1 and COL16A1), a signaling molecule (WNT11), a homeobox homolog (BAPX1), a receptor (IL-1R1), an IGFs modulator (IGFBP5), and a mettaloproteinase (MMP16) increased progressively up to about day 14, suggesting roles later in chondrogenesis. Our results indicate that the simplicity of the system makes it possible to define in detail the cellular and molecular events during chondrogenesis.