Cell type-specific regulation of CCN2 protein expression by PI3K–AKT–FoxO signaling

The biological activity of connective tissue growth factor (CTGF, CCN2) is regulated at the level of intracellular signaling leading to gene expression, and by its extracellular interaction partners which determine the functional outcome of CCN2 action. In this overview, we summarize the data which provide evidence that one of the major signaling pathways, phosphatidylinositol-3 kinase (PI3K)–AKT signaling, shows a remarkable cell type-dependence in terms of regulation of CCN2 expression. In smooth muscle cells, fibroblasts, and epithelial cells, inhibition of this pathway either reduced CCN2 expression or was not involved in CCN2 gene expression depending on the stimulus used. In microvascular endothelial cells by contrast, activation of PI3K–AKT signaling was inversely related to CCN2 expression. Upregulation of CCN2 upon inhibition of PI3K–AKT was also observed in primary cultures of human endothelial cells (HUVEC) exposed to laminar flow in an in vitro flow-through system. In different types of endothelial cells, FoxO transcription factors, which are negatively regulated by AKT, were identified as potent activators of CCN2 gene expression. In HUVEC, we observed a correlation between enhanced nuclear localization of FoxO1 and increased synthesis of CCN2 protein in areas of non-uniform shear stress. These data indicate that FoxO proteins are key regulators of CCN2 gene expression which determine the effect of PI3K–AKT activation in terms of CCN2 regulation. Short summary Phosphatidylinositol-3 kinase (PI3K)–AKT signaling shows a remarkable cell type-dependence in terms of regulation of CCN2 expression. In endothelial cells activation of PI3K - AKT signaling was inversely related to CCN2 expression. FoxO transcription factors, which are negatively regulated by AKT, were identified as potent activators of CCN2 gene expression

ERK1/2 signaling dominates over RhoA signaling in regulating early changes in RNA expression induced by endothelin-1 in neonatal rat cardiomyocytes

Cardiomyocyte hypertrophy is associated with changes in gene expression. Extracellular signal-regulated kinases 1/2 (ERK1/2) and RhoA [activated by hypertrophic agonists (e.g. endothelin-1)] regulate gene expression and are implicated in the response, but their relative significance in regulating the cardiomyocyte transcriptome is unknown. Our aim was to establish the significance of ERK1/2 and/or RhoA in the early cardiomyocyte transcriptomic response to endothelin-1.Cardiomyocytes were exposed to endothelin-1 (1 h) with/without PD184352 (to inhibit ERK1/2) or C3 transferase (C3T, to inhibit RhoA). RNA expression was analyzed using microarrays and qPCR. ERK1/2 signaling positively regulated approximately 65% of the early gene expression response to ET-1 with a small (approximately 2%) negative effect, whereas RhoA signaling positively regulated approximately 10% of the early gene expression response to ET-1 with a greater (approximately 14%) negative contribution. Of RNAs non-responsive to endothelin-1, 66 or 448 were regulated by PD184352 or C3T, respectively, indicating that RhoA had a more significant effect on baseline RNA expression. mRNAs upregulated by endothelin-1 encoded a number of receptor ligands (e.g. Ereg, Areg, Hbegf) and transcription factors (e.g. Abra/Srf) that potentially propagate the response.ERK1/2 dominates over RhoA in the early transcriptomic response to endothelin-1. RhoA plays a major role in maintaining baseline RNA expression but, with upregulation of Abra/Srf by endothelin-1, RhoA may regulate changes in RNA expression over longer times. Our data identify ERK1/2 as a more significant node than RhoA in regulating the early stages of cardiomyocyte hypertrophy

Connective Tissue Growth Factor in Regulation of RhoA Mediated Cytoskeletal Tension Associated Osteogenesis of Mouse Adipose-Derived Stromal Cells

Background: Cytoskeletal tension is an intracellular mechanism through which cells convert a mechanical signal into a biochemical response, including production of cytokines and activation of various signaling pathways. Methods/Principal Findings: Adipose-derived stromal cells (ASCs) were allowed to spread into large cells by seeding them at a low-density (1,250 cells/cm 2), which was observed to induce osteogenesis. Conversely, ASCs seeded at a high-density (25,000 cells/cm 2) featured small cells that promoted adipogenesis. RhoA and actin filaments were altered by changes in cell size. Blocking actin polymerization by Cytochalasin D influenced cytoskeletal tension and differentiation of ASCs. To understand the potential regulatory mechanisms leading to actin cytoskeletal tension, cDNA microarray was performed on large and small ASCs. Connective tissue growth factor (CTGF) was identified as a major regulator of osteogenesis associated with RhoA mediated cytoskeletal tension. Subsequently, knock-down of CTGF by siRNA in ASCs inhibited this osteogenesis. Conclusions/Significance: We conclude that CTGF is important in the regulation of cytoskeletal tension mediated AS

The role of CCN2 in cartilage and bone development

CCN2, a classical member of the CCN family of matricellular proteins, is a key molecule that conducts cartilage development in a harmonized manner through novel molecular actions. During vertebrate development, all cartilage is primarily formed by a process of mesenchymal condensation, while CCN2 is induced to promote this process. Afterwards, cartilage develops into several subtypes with different fates and missions, in which CCN2 plays its proper roles according to the corresponding microenvironments. The history of CCN2 in cartilage and bone began with its re-discovery in the growth cartilage in long bones, which determines the skeletal size through the process of endochondral ossification. CCN2 promotes physiological developmental processes not only in the growth cartilage but also in the other types of cartilages, i.e., Meckel’s cartilage representing temporary cartilage without autocalcification, articular cartilage representing hyaline cartilage with physical stiffness, and auricular cartilage representing elastic cartilage. Together with its significant role in intramembranous ossification, CCN2 is regarded as a conductor of skeletogenesis. During cartilage development, the CCN2 gene is dynamically regulated to yield stage-specific production of CCN2 proteins at both transcriptional and post-transcriptional levels. New functional aspects of known biomolecules have been uncovered during the course of investigating these regulatory systems in chondrocytes. Since CCN2 promotes integrated regeneration as well as generation (=development) of these tissues, its utility in regenerative therapy targeting chondrocytes and osteoblasts is indicated, as has already been supported by experimental evidence obtained in vivo

Gene Bionetwork Analysis of Ovarian Primordial Follicle Development

Ovarian primordial follicles are critical for female reproduction and comprise a finite pool of gametes arrested in development. A systems biology approach was used to identify regulatory gene networks essential for primordial follicle development. Transcriptional responses to eight different growth factors known to influence primordial follicles were used to construct a bionetwork of regulatory genes involved in rat primordial follicle development. Over 1,500 genes were found to be regulated by the various growth factors and a network analysis identified critical gene modules involved in a number of signaling pathways and cellular processes. A set of 55 genes was identified as potential critical regulators of these gene modules, and a sub-network associated with development was determined. Within the network two previously identified regulatory genes were confirmed (i.e., Pdgfa and Fgfr2) and a new factor was identified, connective tissue growth factor (CTGF). CTGF was tested in ovarian organ cultures and found to stimulate primordial follicle development. Therefore, the relevant gene network associated with primordial follicle development was validated and the critical genes and pathways involved in this process were identified. This is one of the first applications of network analysis to a normal developmental process. These observations provide insights into potential therapeutic targets for preventing ovarian disease and promoting female reproduction

Early and Late Postnatal Myocardial and Vascular Changes in a Protein Restriction Rat Model of Intrauterine Growth Restriction

Intrauterine growth restriction (IUGR) is a risk factor for cardiovascular disease in later life. Early structural and functional changes in the cardiovascular system after IUGR may contribute to its pathogenesis. We tested the hypothesis that IUGR leads to primary myocardial and vascular alterations before the onset of hypertension. A rat IUGR model of maternal protein restriction during gestation was used. Dams were fed low protein (LP; casein 8.4%) or isocaloric normal protein diet (NP; casein 17.2%). The offspring was reduced to six males per litter. Immunohistochemical and real-time PCR analyses were performed in myocardial and vascular tissue of neonates and animals at day 70 of life. In the aortas of newborn IUGR rats expression of connective tissue growth factor (CTGF) was induced 3.2-fold. At day 70 of life, the expression of collagen I was increased 5.6-fold in aortas of IUGR rats. In the hearts of neonate IUGR rats, cell proliferation was more prominent compared to controls. At day 70 the expression of osteopontin was induced 7.2-fold. A 3- to 7-fold increase in the expression of the profibrotic cytokines TGF-β and CTGF as well as of microfibrillar matrix molecules was observed. The myocardial expression and deposition of collagens was more prominent in IUGR animals compared to controls at day 70. In the low-protein diet model, IUGR leads to changes in the expression patterns of profibrotic genes and discrete structural abnormalities of vessels and hearts in adolescence, but, with the exception of CTGF, not as early as at the time of birth. Invasive and non-invasive blood pressure measurements confirmed that IUGR rats were normotensive at the time point investigated and that the changes observed occurred independently of an increased blood pressure. Hence, altered matrix composition of the vascular wall and the myocardium may predispose IUGR animals to cardiovascular disease later in life

The Cholesterol Metabolite 25-Hydroxycholesterol Activates Estrogen Receptor α-Mediated Signaling in Cancer Cells and in Cardiomyocytes

The hydroxylated derivatives of cholesterol, such as the oxysterols, play important roles in lipid metabolism. In particular, 25-hydroxycholesterol (25 HC) has been implicated in a variety of metabolic events including cholesterol homeostasis and atherosclerosis. 25 HC is detectable in human plasma after ingestion of a meal rich in oxysterols and following a dietary cholesterol challenge. In addition, the levels of oxysterols, including 25 HC, have been found to be elevated in hypercholesterolemic serum.Here, we demonstrate that the estrogen receptor (ER) α mediates gene expression changes and growth responses induced by 25 HC in breast and ovarian cancer cells. Moreover, 25 HC exhibits the ERα-dependent ability like 17 β-estradiol (E2) to inhibit the up-regulation of HIF-1α and connective tissue growth factor by hypoxic conditions in cardiomyocytes and rat heart preparations and to prevent the hypoxia-induced apoptosis.The estrogen action exerted by 25 HC may be considered as an additional factor involved in the progression of breast and ovarian tumors. Moreover, the estrogen-like activity of 25 HC elicited in the cardiovascular system may play a role against hypoxic environments

DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines

<p>Abstract</p> <p>Background</p> <p>Despite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi) represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes.</p> <p>Methods</p> <p>HCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity^®Pathway Analysis.</p> <p>Results</p> <p>Treatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed.</p> <p>Conclusion</p> <p>This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets that are modulated by HDACi, providing much-needed information on HDACi mechanism of action and providing rationale for novel drug combination partners. We identified a core signature of 11 genes which were modulated by both vorinostat and LBH589 in a similar manner in both cell lines. These core genes will assist in the development and validation of a common gene set which may represent a molecular signature of HDAC inhibition in colon cancer.</p