Methyltransferase G9A Regulates Osteogenesis via Twist Gene Repression.

Here we investigate the role of epigenetic factors in controlling the timing of cranial neural crest cell differentiation. The gene coding for histone H3 lysine 9 methyltransferase G9A was conditionally deleted in neural crest cells with Wnt1-Cre. The majority of homozygous-null animals survived to birth but thereafter failed to thrive. Phenotypic analysis of postnatal animals revealed that the mutants displayed incomplete ossification and 20% shorter jaws as compared to their wild-type littermates. At E13.5, patterns of expression of the osteogenic transcription factor RUNX2 and the mesenchymal transcription factor TWIST are similar in controls and mutants; both overlap in areas of future intramembranous bone formation. At E14.5, the nonosteogenic mesenchyme expressed TWIST, whereas the ossification center had strong RUNX2 and osteopontin expression. In the mutants, TWIST protein was present in the osteogenic mesenchyme, while osteopontin was not expressed until E15.5. In addition, in mutants, small regions of TWIST-positive osteogenic mesenchyme were visible until E15.5. The delay in ossification and reduction in size of the ossification centers were correlated with an earlier decrease in proliferation. We used micromass cultures of the face to investigate the direct effects of G9A inhibition on skeletal differentiation. Addition of a small molecule inhibitor for G9A, BIX-01294, to wild-type cells upregulated Twist genes similar to what was observed in vivo. The inhibitor also caused decreases in several osteogenic markers. Chromatin immunoprecipitation analysis of primary osteogenic mesenchyme from calvaria revealed that Twist1 and Twist2 regulatory regions contain the repressive H3K9me2 marks catalyzed by G9A, which are removed when BIX-01294 is added. Our results establish a role for G9A and H3K9me2 in the regulation of Twist genes and provide novel insights into the significance of epigenetic mechanisms in controlling temporal and tissue-specific gene expression during development.

The transcriptional repressor HIC1 regulates intestinal immune homeostasis.

The intestine is a unique immune environment that must respond to infectious organisms but remain tolerant to commensal microbes and food antigens. However, the molecular mechanisms that regulate immune cell function in the intestine remain unclear. Here we identify the POK/ZBTB family transcription factor hypermethylated in cancer 1 (HIC1, ZBTB29) as a central component of immunity and inflammation in the intestine. HIC1 is specifically expressed in immune cells in the intestinal lamina propria (LP) in the steady state and mice with a T-cell-specific deletion of HIC1 have reduced numbers of T cells in the LP. HIC1 expression is regulated by the Vitamin A metabolite retinoic acid, as mice raised on a Vitamin A-deficient diet lack HIC1-positive cells in the intestine. HIC1-deficient T cells overproduce IL-17A in vitro and in vivo, and fail to induce intestinal inflammation, identifying a critical role for HIC1 in the regulation of T-cell function in the intestinal microenvironment under both homeostatic and inflammatory conditions.

SS18-SSX2 and the mitochondrial apoptosis pathway in mouse and human synovial sarcomas.

Synovial sarcoma is a deadly malignancy with limited sensitivity to traditional cytotoxic chemotherapy. SS18-SSX fusion oncogene expression characterizes human synovial sarcomas and drives oncogenesis in a mouse model. Elevated expression of BCL2 is considered a consistent feature of the synovial sarcoma expression profile. Our objective was to evaluate the expression of apoptotic pathway members in synovial sarcomas and interrogate the impact of modulating SS18-SSX expression on this pathway. We show in human and murine synovial sarcoma cells that SS18-SSX increases BCL2 expression, but represses other anti-apoptotic genes, including MCL1 and BCL2A1. This repression is achieved by directly suppressing expression via binding through activating transcription factor 2 (ATF2) to the cyclic adenosine monophosphate (AMP) response element (CRE) in the promoters of these genes and recruiting TLE1/Groucho. The suppression of these two anti-apoptotic pathways silences the typical routes by which other tumors evade BH3-domain peptidomimetic pharmacotherapy. We show that mouse and human synovial sarcoma cells are sensitive in vitro to ABT-263, a BH3-peptidomimetic, much more than the other tested cancer cell lines. ABT-263 also enhances the sensitivity of these cells to doxorubicin, a traditional cytotoxic chemotherapy used for synovial sarcoma. We also demonstrate the capacity of ABT-263 to stunt synovial sarcomagenesis in vivo in a genetic mouse model. These data recommend pursuit of BH3-peptidomimetic pharmacotherapy in human synovial sarcomas.

Mechanical force modulates scleraxis expression in bioartificial tendons.

Following tendon injury, cartilage, bone and fat metaplasia are often observed, making the optimization of tenocyte differentiation an important clinical goal. In this study we examined the effect of static and cyclic mechanical loading on the expression of genes which play a role in tenocyte differentiation and function, namely scleraxis (Scx) and Type I collagen (Col1a1), and determined the effect of varying mechanical parameters including (1) static vs dynamic load, (2) increasing strain magnitude, (3) inclusion of 10 s rest periods, and (4) increasing cycle number. Cyclic loading resulted in a greater increase of tenocyte gene expression than static loading over 3 weeks in culture. Increasing strain levels potentiated the induction of tenocyte genes. The insertion of a 10 s rest periods further enhanced tenocyte gene expression, as did increasing repetition numbers. These results suggest that mechanical signaling exerts an important influence on the expression of genes which play a role in determining the tendon phenotype. Further work is required to confirm and extend these findings in primary cells such as resident tendon progenitor/stem cells, in order to provide an improved understanding of biology from which optimized rehabilitation programs can be developed.

EGR1 reactivation by histone deacetylase inhibitors promotes synovial sarcoma cell death through the PTEN tumor suppressor.

Synovial sarcoma is a high-grade soft tissue malignancy, for which current cytotoxic chemotherapies provide limited benefit. Although histone deacetylase (HDAC) inhibitors are known to suppress synovial sarcoma in vitro and in vivo, the exact mechanism is not clear. In this study, we report a central role of the transcription factor, early growth response-1 (EGR1), in the regulation of HDAC inhibitor-induced apoptotic cell death in synovial sarcoma. The SS18-SSX oncoprotein, characteristic of synovial sarcoma, maintains EGR1 expression at low levels, whereas it is significantly increased after HDAC inhibitor treatment. On the contrary, EGR1 knockdown leads to a decrease in HDAC inhibitor-induced apoptosis. Moreover, we find that under these conditions phosphatase and tensin homolog deleted in chromosome 10 (PTEN) is upregulated and this occurs through direct binding of EGR1 to an element upstream of the PTEN promoter. Using a combination of gain- and loss-of-function approaches, we show that EGR1 modulation of PTEN contributes to HDAC inhibitor-induced apoptosis in synovial sarcoma. Finally, restoration of EGR1 or PTEN expression is sufficient to induce synovial sarcoma cell death. Taken together, our findings indicate that SS18-SSX-mediated attenuation of an EGR1-PTEN network regulates synovial sarcoma cell survival, and that HDAC inhibitor-mediated apoptosis operates at least in part through reactivation of this pathway.

Induction of collagen mineralization by a bone sialoprotein--decorin chimeric protein.

The observation that hydroxyapatite (HA) formation from metastable solutions can be induced by nucleating proteins such as bone sialoprotein (BSP) suggests a possible treatment for bone defects. The introduction of a mixture of nucleating protein and type I collagen should result in a defect becoming filled with a mineralized collagenous matrix that is biologically and mechanically compatible and capable of being remodeled. To create a nucleating protein that would interact with collagen fibrils, we combined the putative collagen-binding site of mouse decorin with one of two putative HA-nucleating sites of pig BSP. The resulting chimeric protein induced the formation of HA crystals in a steady-state agarose gel system and bound with high affinity to fibrillar type I collagen. The addition of chimeric protein to collagen gels perfused with low concentrations of calcium and phosphate resulted in the deposition of large, apparently needle-shaped HA crystals on the surface of collagen fibrils. These findings suggest that the BSP-decorin chimeric protein could be capable of inducing the mineralization of collagen in vivo.

Retinoid signalling and skeletal development.

Metabolites of vitamin A, including retinoic acid (RA), comprise a class of molecules known to be important in development and homeostasis. RA functions through a class of nuclear hormone receptors, the RA receptors (RARs), to regulate gene transcription. In the developing mammalian limb, RA affects the differentiation of many cell lineages, including those of the chondrogenic lineage. In excess, RA is a potent teratogen, causing characteristic skeletal defects in a stage- and dose-dependent manner. Genetic analysis has shown that the absence of RARs leads to severe deficiencies in cartilage formation at certain anatomical locations while promoting ectopic cartilage formation at other sites. Expression of either a dominant-negative or a weak constitutively active RAR in the developing limbs of transgenic mice adversely affects chondrogenesis leading to skeletal malformations. Together, these results show that RAR-mediated signalling plays a fundamental role in skeletogenesis. This chapter will focus on the function of RARs in regulating chondroblast differentiation and the contribution of RA signalling to appositional and longitudinal growth of the skeletal primordia.

Functional analysis of bone sialoprotein: identification of the hydroxyapatite-nucleating and cell-binding domains by recombinant peptide expression and site-directed mutagenesis.

Mammalian bone sialoprotein (BSP) is a mineralized tissue-specific protein containing an RGD (arginine-glycine-aspartic acid) cell-attachment sequence and two distinct glutamic acid (glu)-rich regions, with each containing one contiguous glu sequence. These regions have been proposed to contribute to the attachment of bone cells to the extracellular matrix and to the nucleation of hydroxyapatite (HA), respectively. To further delineate the domains responsible for these activities, porcine BSP cDNA was used to construct expression vectors coding for two partial-length recombinant BSP peptides: P2S (residues 42-87), containing the first glutamic acid-rich domain; and P1L (residues 69-300), containing the second glutamic acid-rich region and the RGD sequence. These peptides were expressed in Escherichia coli as his-tag fusion proteins and purified by nickel affinity columns and FPLC chromatography. Digestion with trypsin released the his-tag fusion peptide, which generated P2S-TY (residues 42-87) and P1L-TY (residues 132-239). Using a steady-state agarose gel system, P2S-TY promoted HA nucleation, whereas P2S, P1L, and P1L-TY did not. This implies that the minimum requirement for nucleation of HA resides within the amino acid sequence of the first glutamic acid-rich domain, whereas the second glutamic acid-rich domain may require posttranslational modifications for activity. P1L, but not P2S, promoted RGD-mediated attachment of human gingival fibroblasts in a manner similar to that of native BSP. Deletion of the RGD domain or conversion of it to RGE (arginine-glycine-glutamic acid) abolished the cell-attachment activity of P1L. This suggests that, at least for human gingival fibroblasts, the major cell-attachment activity in the recombinant BSP peptides studied (residues 42-87 and 69-300) requires the RGD sequence located at the C-terminal domain.

Analysis of Nedd4 expression during skeletal development in the mouse limb.

Nedd4, a ubiquitin-protein ligase, was originally identified as being down-regulated during development of the mouse brain (Nedd denotes neural precursor cell expressed developmentally down-regulated) (Kumar, S., Tomooka, Y., Noda, M., 1992. Identification of a set of genes with developmentally down-regulated expression in the mouse brain. Biochem. Biophys. Res. Commun. 185, 1155-1161). Subtractive hybridization was used in an attempt to identify genes that are preferentially expressed early in skeletogenesis. Using this technique Nedd4 was identified multiple times. Northern blot analysis confirmed that Nedd4 is down-regulated in the forelimb and hind limb. In situ hybridization was carried out to identify regions of the limb bud expressing Nedd4. Nedd4 is expressed weakly in condensing mesenchyme, and abundantly in proliferating and prehypertrophic chondrocytes, but is undetectable in hypertrophic chondrocytes. Primary cultures, which closely mimic in vivo chondrogenesis, were also used to demonstrate the stage-specific expression of Nedd4 during early skeletal development.

Regulation of skeletal progenitor differentiation by the BMP and retinoid signaling pathways.

The generation of the paraxial skeleton requires that commitment and differentiation of skeletal progenitors is precisely coordinated during limb outgrowth. Several signaling molecules have been identified that are important in specifying the pattern of these skeletal primordia. Very little is known, however, about the mechanisms regulating the differentiation of limb mesenchyme into chondrocytes. Overexpression of RARalpha in transgenic animals interferes with chondrogenesis and leads to appendicular skeletal defects (Cash, D.E., C.B. Bock, K. Schughart, E. Linney, and T.M. Underhill. 1997. J. Cell Biol. 136:445-457). Further analysis of these animals shows that expression of the transgene in chondroprogenitors maintains a prechondrogenic phenotype and prevents chondroblast differentiation even in the presence of BMPs, which are known stimulators of cartilage formation. Moreover, an RAR antagonist accelerates chondroblast differentiation as demonstrated by the emergence of collagen type II-expressing cells much earlier than in control or BMP-treated cultures. Addition of Noggin to limb mesenchyme cultures inhibits cartilage formation and the appearance of precartilaginous condensations. In contrast, abrogation of retinoid signaling is sufficient to induce the expression of the chondroblastic phenotype in the presence of Noggin. These findings show that BMP and RAR-signaling pathways appear to operate independently to coordinate skeletal development, and that retinoid signaling can function in a BMP-independent manner to induce cartilage formation. Thus, retinoid signaling appears to play a novel and unexpected role in skeletogenesis by regulating the emergence of chondroblasts from skeletal progenitors.

P2X(4) purinoceptors mediate an ATP-activated, non-selective cation current in rabbit osteoclasts.

Extracellular nucleotides act as signaling molecules in numerous tissues. In bone, nucleotides stimulate osteoclast formation and activity; however, the receptors and signaling mechanisms underlying these effects have yet to be identified. To identify specific P2X purinoceptor subtypes in osteoclasts, degenerate oligonucleotide primers were used to PCR-amplify DNA fragments from a rabbit osteoclast cDNA library. A 372-base-pair fragment was obtained that encoded an amino acid sequence with 88% identity to the rat P2X(4) purinoceptor. The presence of P2X(4) mRNA in purified osteoclasts was confirmed by reverse transcription-PCR. Endogenous purinoceptors were functionally characterized in isolated rabbit osteoclasts by patch-clamp recording in whole-cell configuration. At negative membrane potentials, application of ATP or ADP rapidly activated an inward current followed by an outward current. In contrast, UTP or ADPbetaS elicited only an outward current, due to activation of a Ca(2+)-dependent K(+) conductance. The initial inward current was non-selective for cations and inactivated during agonist application. Furthermore, the inward current was insensitive to suramin and Cibacron blue, and was potentiated by Zn(2+). These characteristics are consistent with properties of P2X(4) purinoceptors. Activation of P2X(4) purinoceptors leads to cation influx and depolarization. Nucleotides, released at sites of trauma or inflammation, may act through these receptors on osteoclasts to stimulate bone resorption.

The effects of gap junction blockage on neuronal differentiation of human NTera2/clone D1 cells.

Gap junctions are intercellular channels which provide for the passage of small ions and molecules (MW <1200 D) among adjacent cells. The NTera2/clone D1 (NT2/D1) cells are CNS precursors which differentiate into NT2-N neurons upon treatment with retinoic acid (RA) and antiproliferative agents. In this study, the effects of gap junction blockers 18 alpha-glycyrrhetinic acid (GRA) and carbenoxolone (CBX) have been compared with those of oleanolic acid (OLA) and glycyrrhizic acid (GZA), GRA analogs with no blocking effects. Both control and experimental cultures showed reduction of Cx43 protein after 4 weeks of RA induction. A major reduction was also observed in expression of cytokeratin, vimentin, and nestin in control cells at this time point while the cultures treated with the blockers did not show any significant change. The average number of MAP2-positive NT2-N differentiated neurons per field of view in the cultures treated with the blockers was less than 7% of that of control cultures. NT2-N cells were negative for Cx43, cytokeratin, vimentin, and nestin. The blockers did not appear to be operating through inhibition of RA signaling, as their presence did not affect the expression of retinoic acid receptors (RARalpha and RARgamma) nor did they inhibit RA-mediated gene transcription. These results, together, show that the blockage of gap junctions interferes with neuronal differentiation of NT2/D1 cells.

Gap junction blockage interferes with neuronal and astroglial differentiation of mouse P19 embryonal carcinoma cells.

During embryonic development, cells not only increase in number, they also undergo specialization and differentiate into diverse cell types that are organized into different tissues and organs. Nervous system development, for example, involves a complex series of events such as neuronal and astroglial differentiation that are coordinated among adjacent cells. The organization of growth and differentiation may be mediated, at least partly, by exchange of small ions and molecules via intercellular gap junction channels. These structures are mode of connexons (hemichannels), which are hexameric assemblies of the gap junction proteins, connexins. We investigated the role of intercellular communication in neuronal and astroglial differentiation by using a gap junction blocking agent, carbenoxolone (CBX), in comparison to its inactive (control) analog, glycyrrhizic acid (GZA). We used the mouse P19 embryonal carcinoma cell line, which differentiates into neurons and astrocytes upon retinoic acid (RA) induction. Our results show that both GZA- and CBX-treated cells express alpha 1 connexin (connexin43). The level of alpha 1 connexin decreases upon RA induction. CBX treated cells show significant reduction in both neuronal (5-fold) and astrocytic (13-fold) differentiation compared with those of control. These results clearly indicate that the blockage of gap junction-mediated intercellular communication interferes with differentiation of P19 cells into neurons and astrocytes.

Retinoids and their receptors in skeletal development.

The embryonic vertebrate limb serves as an excellent experimental model system in which to study mechanisms that regulate morphogenesis of the skeleton. The appendicular skeleton arises through the process of endochondral ossification, whereby a cartilage template is initially formed and subsequently replaced by bone. One molecule that has a dramatic effect on these processes is the vitamin-A metabolite, retinoic acid (RA). RA functions through a class of nuclear hormone receptors, the retinoic acid receptors (RARs) and retinoid-X-receptors (RXRs), to regulate gene transcription. Experimental evidence from RA teratogenesis suggests that the presence of ligand-activated RARs and/or inappropriate expression of RARs inhibits chondrogenesis. Conversely, genetic analysis has shown that the absence of the receptors can lead to deficiencies in cartilage formation while also promoting chondrogenesis at ectopic sites. Taken together, these studies suggest that the RARs play a fundamental role in the early stages of skeletal development, specifically those involved in the formation of prechondrogenic condensations and their subsequent differentiation into chondroblasts.

Retinoic acid receptor alpha function in vertebrate limb skeletogenesis: a modulator of chondrogenesis.

Retinoic acid is a signaling molecule involved in the regulation of growth and morphogenesis during development. There are three types of nuclear receptors for all-trans retinoic acid in mammals, RAR alpha, RAR beta, and RAR gamma, which transduce the retinoic acid signal by inducing or repressing the transcription of target genes (Leid, M., P. Kastner, and P. Chambon. 1992. Trends Biochem. Sci. 17:427-433). While RAR alpha, RAR beta, and RAR gamma are expressed in distinct but overlapping patterns in the developing mouse limb, their exact role in limb development remains unclear. To better understand the role of retinoic acid receptors in mammalian limb development, we have ectopically expressed a modified RAR alpha with constitutive activity (Balkan, W., G.K. Klintworth, C.B. Bock, and E. Linney. 1992. Dev. Biol. 151:622-625) in the limbs of transgenic mice. Overexpression of the transgene was associated with marked pre- and postaxial limb defects, particularly in the hind limb, where expression of the transgene was consistently seen across the whole anteroposterior axis. The defects displayed in these mice recapitulate, to a large degree, many of the congenital limb malformations observed in the fetuses of dams administered high doses of retinoic acid (Kochhar, D.M. 1973. Teratology. 7:289-295). Further analysis of these transgenic animals showed that the defect in skeletogenesis resided at the level of chondrogenesis. Comparison of the expression of the transgene relative to that of endogenous RAR alpha revealed that downregulation of RAR alpha is important in allowing the chondrogenic phenotype to be expressed. These results demonstrate a specific function for RARalpha in limb development and the regulation of chondroblast differentiation.

Dialkyldithiocarbamates inhibit tyrosine hydroxylase activity in PC12 cells and in fibroblasts that express tyrosine hydroxylase.

Dithiocarbamates and CS2 have been associated with neurobehavioural changes suggestive of central dopaminergic dysfunction. Diethyldithiocarbamate (DEDC), dimethyldithiocarbamate (DMDC), and methyldithiocarbamate (MDC) were examined for their ability to inhibit tyrosine hydroxylase (TH) activity in PC12 cells and transfected CHO fibroblasts that expressed TH (CHO/TH) activity when tetrahydrobiopterin (BH4) was added to medium. DEDC or DMDC did not significantly alter viability of PC12 cells or CHO/TH cells at < or = 100 microM for 18 h; the EC50 for each compound was approximately 5 mM in both cell lines. In contrast, the EC50 for MDC was 41 or 74 microM in PC12 or CHO/TH cultures, respectively. There was no change in immunodetectable levels of TH in PC12 or CHO/TH cells following exposure to subcytotoxic concentrations of dithiocarbamates. DEDC and DMDC (5 to 100 microM) produced concentration-dependent reductions in PC12 cell dopamine and dopac levels as well as in dopa levels in CHO/TH cultures. Reduction of PC12 catechols was not due to altered vesicular storage. In vitro PC12 TH activity was 80.2 +/- 3.4% or 82.4 +/- 2.9% of control following exposure to 100 microM DEDC or DMDC, respectively, and was not fully restored by incubation with Fe2+. These results show that DEDC and DMDC, but not MDC, are low potency cytotoxins that decrease TH activity in cultured cells through mechanisms other than inhibition of BH4 biosynthesis or iron chelation.

Fibroblasts that express aromatic amino acid decarboxylase have increased sensitivity to the synergistic cytotoxicity of L-dopa and manganese.

Manganism, a neurodegenerative disease that can follow chronic exposure to Mn, has been associated with lesions in the basal ganglia and depletion of dopamine and its metabolites in this brain region. Herein, we have tested the hypothesis that oxidation of catechols is a critical component of Mn-induced cytotoxicity. To eliminate confounding metabolic pathways, a nonneuronal cell line, Chinese hamster ovary (CHO) fibroblasts, was transfected with a cDNA for bovine aromatic amino acid decarboxylase, and a high expressing clone was isolated (CHO/AADC). Exposure of wild-type (CHO/WT) or CHO/AADC cultures to L-dopa (62 to 500 microM) resulted in intracellular accumulation of L-dopa or L-dopa and dopamine, respectively, that was concentration-dependent. Intracellular catechol levels in CHO/AADC cells were double those in CHO/WT cultures. No dopac was identified intra- or extracellularly. Addition of MnCl2 (125 to 500 microM) resulted in cytotoxicity that progressed with increasing concentrations of L-dopa or Mn. Neither L-dopa nor MnCl2 alone was toxic at these concentrations, and cytotoxicity was completely abrogated by substitution of L-tyrosine for L-dopa. Although CHO/AADC cultures were more sensitive than CHO/WT to L-dopa and Mn, this was completely accounted for by the differences in intracellular catechol levels between the two cell lines. Preformed melanin or dopac were low-potency cytotoxins only at high MnCl2 concentrations. These results indicate that Mn and intracellular L-dopa and dopamine, but not extracellular dopac or melanin, are potent synergistic cytotoxins.