Wnt16 attenuates TGFß-induced chondrogenic transformation in vascular smooth muscle.

OBJECTIVE: Phenotypic plasticity of vascular smooth muscle cells (VSMCs) contributes to cardiovascular disease. Chondrocyte-like transformation of VSMCs associates with vascular calcification and underlies the formation of aortic cartilaginous metaplasia induced in mice by genetic loss of matrix Gla protein (MGP). Previous microarray analysis identified a dramatic downregulation of Wnt16 in calcified MGP-null aortae, suggesting an antagonistic role for Wnt16 in the chondrogenic transformation of VSMCs. APPROACH AND RESULTS: Wnt16 is significantly downregulated in MGP-null aortae, before the histological appearance of cartilaginous metaplasia, and in primary MGP-null VSMCs. In contrast, intrinsic TGFß is activated in MGP-null VSMCs and is necessary for spontaneous chondrogenesis of these cells in high-density micromass cultures. TGFß3-induced chondrogenic transformation in wild-type VSMCs associates with Smad2/3-dependent Wnt16 downregulation, but Wnt16 does not suppress TGFß3-induced Smad activation. In addition, TGFß3 inhibits Notch signaling in wild-type VSMCs, and this pathway is downregulated in MGP-null aortae. Exogenous Wnt16 stimulates Notch activity and attenuates TGFß3-induced downregulation of Notch in wild-type VSMCs, prevents chondrogenesis in MGP-null and TGFß3-treated wild-type VSMCs, and stabilizes expression of contractile markers of differentiated VSMCs. CONCLUSIONS: We describe a novel TGFß-Wnt16-Notch signaling conduit in the chondrocyte-like transformation of VSMCs and identify endogenous TGFß activity in MGP-null VSMCs as a critical mediator of chondrogenesis. Our proposed model suggests that the activated TGFß pathway inhibits expression of Wnt16, which is a positive regulator of Notch signaling and a stabilizer of VSMC phenotype. These data advance the comprehensive mechanistic understanding of VSMC transformation and may identify a novel potential therapeutic target in vascular calcification.

Two sides of MGP null arterial disease: chondrogenic lesions dependent on transglutaminase 2 and elastin fragmentation associated with induction of adipsin.

Mutations in matrix Gla protein (MGP) have been correlated with vascular calcification. In the mouse model, MGP null vascular disease presents as calcifying cartilaginous lesions and mineral deposition along elastin lamellae (elastocalcinosis). Here we examined the mechanisms underlying both of these manifestations. Genetic ablation of enzyme transglutaminase 2 (TG2) in Mgp(-/-) mice dramatically reduced the size of cartilaginous lesions in the aortic media, attenuated calcium accrual more than 2-fold, and doubled longevity as compared with control Mgp(-/-) animals. Nonetheless, the Mgp(-/-);Tgm2(-/-) mice still died prematurely as compared with wild-type and retained the elastocalcinosis phenotype. This pathology in Mgp(-/-) animals was developmentally preceded by extensive fragmentation of elastic lamellae and associated with elevated serine elastase activity in aortic tissue and vascular smooth muscle cells. Systematic gene expression analysis followed by an immunoprecipitation study identified adipsin as the major elastase that is induced in the Mgp(-/-) vascular smooth muscle even in the TG2 null background. These results reveal a central role for TG2 in chondrogenic transformation of vascular smooth muscle and implicate adipsin in elastin fragmentation and ensuing elastocalcinosis. The importance of elastin calcification in MGP null vascular disease is highlighted by significant residual vascular calcification and mortality in Mgp(-/-);Tgm2(-/-) mice with reduced cartilaginous lesions. Our studies identify two potential therapeutic targets in vascular calcification associated with MGP dysfunction and emphasize the need for a comprehensive approach to this multifaceted disorder.

Map of open and closed chromatin domains in Drosophila genome.

BACKGROUND: Chromatin compactness has been considered a major determinant of gene activity and has been associated with specific chromatin modifications in studies on a few individual genetic loci. At the same time, genome-wide patterns of open and closed chromatin have been understudied, and are at present largely predicted from chromatin modification and gene expression data. However the universal applicability of such predictions is not self-evident, and requires experimental verification. RESULTS: We developed and implemented a high-throughput analysis for general chromatin sensitivity to DNase I which provides a comprehensive epigenomic assessment in a single assay. Contiguous domains of open and closed chromatin were identified by computational analysis of the data, and correlated to other genome annotations including predicted chromatin "states", individual chromatin modifications, nuclear lamina interactions, and gene expression. While showing that the widely trusted predictions of chromatin structure are correct in the majority of cases, we detected diverse "exceptions" from the conventional rules. We found a profound paucity of chromatin modifications in a major fraction of closed chromatin, and identified a number of loci where chromatin configuration is opposite to that expected from modification and gene expression patterns. Further, we observed that chromatin of large introns tends to be closed even when the genes are expressed, and that a significant proportion of active genes including their promoters are located in closed chromatin. CONCLUSIONS: These findings reveal limitations of the existing predictive models, indicate novel mechanisms of epigenetic regulation, and provide important insights into genome organization and function.

Transglutaminase inhibitors attenuate vascular calcification in a preclinical model.

OBJECTIVE: In vitro, transglutaminase-2 (TG2)-mediated activation of the ß-catenin signaling pathway is central in warfarin-induced calcification, warranting inquiry into the importance of this signaling axis as a target for preventive therapy of vascular calcification in vivo. METHODS AND RESULTS: The adverse effects of warfarin-induced elastocalcinosis in a rat model include calcification of the aortic media, loss of the cellular component in the vessel wall, and isolated systolic hypertension, associated with accumulation and activation of TG2 and activation of ß-catenin signaling. These effects of warfarin can be completely reversed by intraperitoneal administration of the TG2-specific inhibitor KCC-009 or dietary supplementation with the bioflavonoid quercetin, known to inhibit ß-catenin signaling. Our study also uncovers a previously uncharacterized ability of quercetin to inhibit TG2. Quercetin reversed the warfarin-induced increase in systolic pressure, underlying the functional consequence of this treatment. Molecular analysis shows that quercetin diet stabilizes the phenotype of smooth muscle and prevents its transformation into osteoblastic cells. CONCLUSIONS: Inhibition of the TG2/ß-catenin signaling axis seems to prevent warfarin-induced elastocalcinosis and to control isolated systolic hypertension.

The nuclear lamina as a gene-silencing hub.

There is accumulating evidence that the nuclear periphery is a transcriptionally repressive compartment. A surprisingly large fraction of the genome is either in transient or permanent contact with nuclear envelope, where the majority of genes are maintained in a silent state, waiting to be awakened during cell differentiation. The integrity of the nuclear lamina and the histone deacetylase activity appear to be essential for gene repression at the nuclear periphery. However, the molecular mechanisms of silencing, as well as the events that lead to the activation of lamina-tethered genes, require further elucidation. This review summarizes recent advances in understanding of the mechanisms that link nuclear architecture, local chromatin structure, and gene regulation.

Lack of global meiotic sex chromosome inactivation, and paucity of tissue-specific gene expression on the Drosophila X chromosome.

BACKGROUND: Paucity of male-biased genes on the Drosophila X chromosome is a well-established phenomenon, thought to be specifically linked to the role of these genes in reproduction and/or their expression in the meiotic male germline. In particular, meiotic sex chromosome inactivation (MSCI) has been widely considered a driving force behind depletion of spermatocyte-biased X-linked genes in Drosophila by analogy with mammals, even though the existence of global MCSI in Drosophila has not been proven. RESULTS: Microarray-based study and qRT-PCR analyses show that the dynamics of gene expression during testis development are very similar between X-linked and autosomal genes, with both showing transcriptional activation concomitant with meiosis. However, the genes showing at least ten-fold expression bias toward testis are significantly underrepresented on the X chromosome. Intriguingly, the genes with similar expression bias toward tissues other than testis, even those not apparently associated with reproduction, are also strongly underrepresented on the X. Bioinformatics analysis shows that while tissue-specific genes often bind silencing-associated factors in embryonic and cultured cells, this trend is less prominent for the X-linked genes. CONCLUSIONS: Our data show that the global meiotic inactivation of the X chromosome does not occur in Drosophila. Paucity of testis-biased genes on the X appears not to be linked to reproduction or germline-specific events, but rather reflects a general underrepresentation of tissue-biased genes on this chromosome. Our analyses suggest that the activation/repression switch mechanisms that probably orchestrate the highly-biased expression of tissue-specific genes are generally not efficient on the X chromosome. This effect, probably caused by dosage compensation counteracting repression of the X-linked genes, may be the cause of the exodus of highly tissue-biased genes to the autosomes.

Analysis of the Drosophila melanogaster testes transcriptome reveals coordinate regulation of paralogous genes.

Gene duplications have been broadly implicated in the generation of testis-specific genes. To perform a comprehensive analysis of paralogous testis-biased genes, we characterized the testes transcriptome of Drosophila melanogaster by comparing gene expression in testes vs. ovaries, heads, and gonadectomized males. A number of the identified 399 testis-biased genes code for the known components of mature sperm. Among the detected 69 genes downregulated in testes, a large fraction is required for viability. By analyzing paralogs of testis-biased genes, we identified "co-regulated" paralogous pairs in which both genes are testis biased, "anti-regulated" pairs in which one paralog is testis biased and the other downregulated in testes, and "neutral" pairs in which one paralog is testis biased and the other constitutively expressed. The numbers of identified co-regulated and anti-regulated pairs were higher than expected by chance. Testis-biased genes included in these pairs show decreased frequency of lethal mutations, suggesting their specific role in male reproduction. These genes also show exceptionally high interspecific variability of expression in comparison between D. melanogaster and the closely related D. simulans. Further, interspecific changes in testis bias of expression are generally correlated within the co-regulated pairs and are anti-correlated within the anti-regulated pairs, suggesting coordinated regulation within both types of paralogous gene pairs.

The pattern of chromosome folding in interphase is outlined by the linear gene density profile.

Spatial organization of chromatin in the interphase nucleus plays a role in gene expression and inheritance. Although it appears not to be random, the principles of this organization are largely unknown. In this work, we show an explicit relationship between the intranuclear localization of various chromosome segments and the pattern of gene distribution along the genome sequence. Using a 7-megabase-long region of the Drosophila melanogaster chromosome 2 as a model, we observed that the six gene-poor chromosome segments identified in the region interact with components of the nuclear matrix to form a compact stable cluster. The six gene-rich segments form a spatially segregated unstable cluster dependent on nonmatrix nuclear proteins. The resulting composite structure formed by clusters of gene-rich and gene-poor regions is reproducible between the nuclei. We suggest that certain aspects of chromosome folding in interphase are predetermined and can be inferred through in silico analysis of chromosome sequence, using gene density profile as a manifestation of "folding code."

Evidence for a hierarchical transcriptional circuit in Drosophila male germline involving testis-specific TAF and two gene-specific transcription factors, Mod and Acj6.

To analyze transcription factors involved in gene regulation by testis-specific TAF (tTAF), tTAF-dependent promoters were mapped and analyzed in silico. Core promoters show decreased AT content, paucity of classical promoter motifs, and enrichment with translation control element CAAAATTY. Scanning of putative regulatory regions for known position frequency matrices identified 19 transcription regulators possibly contributing to tTAF-driven gene expression. Decreased male fertility associated with mutation in one of the regulators, Acj6, indicates its involvement in male reproduction. Transcriptome study of testes from male mutants for tTAF, Acj6, and previously characterized tTAF-interacting factor Modulo implies the existence of a regulatory hierarchy of tTAF, Modulo and Acj6, in which Modulo and/or Acj6 regulate one-third of tTAF-dependent genes.

Up-regulation of the Ku heterodimer in Drosophila testicular cyst cells.

In Drosophila, developing germline cysts in testis are enveloped by two somatic cyst cells essential for germline development and male reproduction. The cyst cells continue development along with the germline. However, the mechanisms of somatic gene expression in testes are poorly understood. We report transcriptional up-regulation of the Ku heterodimer in cyst cells. The initial up-regulation is independent of germline, and transcription is further augmented during spermatogenesis. Abundance of Ku in the cyst cell cytoplasm suggests the role for Ku subunits in the regulation of sperm individualization.

Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster.

Recently, the phenomenon of clustering of co-expressed genes on chromosomes was discovered in eukaryotes. To explore the hypothesis that genes within clusters occupy shared chromatin domains, we performed a detailed analysis of transcription pattern and chromatin structure of a cluster of co-expressed genes. We found that five non-homologous genes (Crtp, Yu, CK2betates, Pros28.1B and CG13581) are expressed exclusively in Drosophila melanogaster male germ-line and form a non-interrupted cluster in the 15 kb region of chromosome 2. The cluster is surrounded by genes with broader transcription patterns. Analysis of DNase I sensitivity revealed 'open' chromatin conformation in the cluster and adjacent regions in the male germ-line cells, where all studied genes are transcribed. In contrast, in somatic tissues where the cluster genes are silent, the domain of repressed chromatin encompassed four out of five cluster genes and an adjacent non-cluster gene CG13589 that is also silent in analyzed somatic tissues. The fifth cluster gene (CG13581) appears to be excluded from the chromatin domain occupied by the other four genes. Our results suggest that extensive clustering of co-expressed genes in eukaryotic genomes does in general reflect the domain organization of chromatin, although domain borders may not exactly correspond to the margins of gene clusters.

Phylogenetic shadowing of a histamine-gated chloride channel involved in insect vision.

A recently identified gene, hclA (synonym: ort), codes for an ionotrophic histamine receptor subunit in Drosophila melanogaster, and known hclA mutations lead to defects in the visual system, neurologic disorders and changed responsiveness to neurotoxins. To investigate whether this novel class of receptors is common across the Insecta, we analysed the genomes of 15 other insect species (Diptera, Hymenoptera, Coleoptera, Lepidoptera) and revealed orthologs of hclA in all of them. The predicted receptor domain of HCLA is extensively conserved (86-100% of identity) among the 16 proteins. Minor changes in the amino acid sequence that includes the putative transmembrane domains (TMs) 1-3 were found in non-drosophilid species only. Substantial amino acid variability was observed in the signal polypeptides, the intracellular loop domains and in TM4, in good accordance with known data on sequence variations in ligand-gated ion channels. Pairwise comparisons revealed three consensus sequences for N-glycosylation, conserved in HCLAs of all species studied, as well as a drosophilid-specific putative phosphorylation site. Real-time PCR analysis demonstrated that hclA-mRNA is abundant in heads of adult Drosophila. However, species- and sex-specific variations of the hclA expression levels were also observed.

Unexpected stability of mariner transgenes in Drosophila.

A number of mariner transformation vectors based on the mauritiana subfamily of transposable elements were introduced into the genome of Drosophila melanogaster and examined for their ability to be mobilized by the mariner transposase. Simple insertion vectors were constructed from single mariner elements into which exogenous DNA ranging in size from 1.3 to 4.5 kb had been inserted; composite vectors were constructed with partial or complete duplications of mariner flanking the exogenous DNA. All of the simple insertion vectors showed levels of somatic and germline excision that were at least 100-fold lower than the baseline level of uninterrupted mariner elements. Although composite vectors with inverted duplications were unable to be mobilized at detectable frequencies, vectors with large direct duplications of mariner could be mobilized. A vector consisting of two virtually complete elements flanking exogenous DNA yielded a frequency of somatic eye-color mosaicism of approximately 10% and a frequency of germline excision of 0.04%. These values are far smaller than those observed for uninterrupted elements. The results imply that efficient mobilization of mariner in vivo requires the presence and proper spacing of sequences internal to the element as well as the inverted repeats.

Role of histone deacetylases in gene regulation at nuclear lamina.

Theoretical models suggest that gene silencing at the nuclear periphery may involve "closing" of chromatin by transcriptional repressors, such as histone deacetylases (HDACs). Here we provide experimental evidence confirming these predictions. Histone acetylation, chromatin compactness, and gene repression in lamina-interacting multigenic chromatin domains were analyzed in Drosophila S2 cells in which B-type lamin, diverse HDACs, and lamina-associated proteins were downregulated by dsRNA. Lamin depletion resulted in decreased compactness of the repressed multigenic domain associated with its detachment from the lamina and enhanced histone acetylation. Our data reveal the major role for HDAC1 in mediating deacetylation, chromatin compaction, and gene silencing in the multigenic domain, and an auxiliary role for HDAC3 that is required for retention of the domain at the lamina. These findings demonstrate the manifold and central involvement of class I HDACs in regulation of lamina-associated genes, illuminating a mechanism by which these enzymes can orchestrate normal and pathological development.

Transglutaminase 2 regulates early chondrogenesis and glycosaminoglycan synthesis.

The expression pattern for tissue transglutaminase (TG2) suggests that it regulates cartilage formation. We analyzed the role of TG2 in early stages of chondrogenesis using differentiating high-density cultures of mesenchymal cells from chicken limb bud as a model. We demonstrate that TG2 promotes cell differentiation towards a pre-hypertrophic stage without inducing precocious hypertrophic maturation. This finding, combined with distinctive up-regulation of extracellular TG2 in the pre-hypertrophic cartilage of the growth plate, indicates that TG2 is an autocrine regulator of chondrocyte differentiation. We also show that TG2 regulates synthesis of the cartilaginous glycosaminoglycan (GAG)-rich extracellular matrix. Elevated levels of TG2 down-regulate xylosyltransferase activity which mediates the key steps in chondroitin sulfate synthesis. On the contrary, inhibition of endogenous transglutaminase activity in differentiating chondrogenic micromasses results in increased GAG deposition and enhancement of early chondrogenic markers. Regulation of GAG synthesis by TG2 appears independent of TGF-ß activity, which is a downstream mediator of the TG2 functions in some biological systems. Instead, our data suggest a major role for cAMP/PKA signaling in transmitting TG2 signals in early chondrogenic differentiation. In summary, we demonstrate that matrix synthesis and early stages of chondrogenic differentiation are regulated through a novel mechanism involving TG2-dependent inhibition of PKA. These findings further advance understanding of cartilage formation and disease, and contribute to cartilage bioengineering.

Nuclear ferritin: a ferritoid-ferritin complex in corneal epithelial cells.

PURPOSE: Ferritin is an iron storage protein that is generally cytoplasmic. However, in embryonic avian corneal epithelial (CE) cells, the authors previously observed that the ferritin was predominantly nuclear. They also obtained evidence that this ferritin protects DNA from oxidative damage by UV light and hydrogen peroxide and that the nuclear localization involves a tissue-specific nuclear transporter, termed ferritoid. In the present investigation, the authors have determined additional properties of the nuclear ferritoid-ferritin complexes. METHODS: For biochemical characterization, a combination of molecular sieve chromatography, immunoblotting, and nuclear-cytoplasmic fractionation was used; DNA binding was analyzed by electrophoretic mobility shift assay. RESULTS: The CE nuclear ferritin complex has characteristics that differentiate it from a "typical" cytoplasmic ferritin, including the presence of ferritin and ferritoid subunits; a molecular weight of approximately 260 kDa, which is approximately half that of cytoplasmic ferritin; its iron content, which is below our limits of detection; and its ability to bind to DNA. CONCLUSIONS: Within CE cell nuclei, ferritin and ferritoid are coassembled into stable complex(es) present in embryonic and adult corneas. Thus, ferritoid not only serves transiently as a nuclear transporter for ferritin, it remains as a component of a unique ferritoid-ferritin nuclear complex.

Regulation of chondrocyte differentiation by actin-severing protein adseverin.

The importance of actin organization in controlling the chondrocyte phenotype is well established, but little is known about the cytoskeletal components regulating chondrocyte differentiation. Previously, we have observed up-regulation of an actin-binding gelsolin-like protein in hypertrophic chondrocytes. We have now identified it as adseverin (scinderin). Adseverin is drastically up-regulated during chondrocyte maturation, as shown by Northern blot analysis, in situ hybridization, and real-time RT-PCR. Its expression is positively regulated by PKC and MEK signaling as shown by inhibitory analyses. Over-expression of adseverin in non-hypertrophic chondrocytes causes rearrangement of the actin cytoskeleton, a change in cell morphology, a dramatic (3.5-fold) increase in cell volume, and up-regulation of Indian hedgehog (Ihh) and of collagen type X--all indicative of chondrocyte differentiation. These changes are mediated by ERK1/2 and p38 kinase pathways. Thus, adseverin-induced rearrangements of the actin cytoskeleton may mediate the PKC-dependent activation of p38 and Erk1/2 signaling pathways necessary for chondrocyte hypertrophy, as evidenced by changes in cell morphology, increase in cell size and expression of the chondrocyte maturation markers. These results demonstrate that interdependence of cytoskeletal organization and chondrogenic gene expression is regulated, at least in part, by actin-binding proteins such as adseverin.

Transcriptional regulation by Modulo integrates meiosis and spermatid differentiation in male germ line.

Transcriptional activation in early spermatocytes involves hundreds of genes, many of which are required for meiosis and spermatid differentiation. A number of the meiotic-arrest genes have been identified as general regulators of transcription; however, the gene-specific transcription factors have remained elusive. To identify such factors, we purified the protein that specifically binds to the promoter of spermatid-differentiation gene Sdic and identified it as Modulo, the Drosophila homologue of nucleolin. Analysis of gene-expression patterns in the male sterile modulo mutant indicates that Modulo supports high expression of the meiotic-arrest genes and is essential for transcription of spermatid-differentiation genes. Expression of Modulo itself is under the control of meiotic-arrest genes and requires the DAZ/DAZL homologue Boule that is involved in the control of G(2)/M transition. Thus, regulatory interactions among Modulo, Boule, and the meiotic-arrest genes integrate meiosis and spermatid differentiation in the male germ line.

Origin and evolution of a new gene expressed in the Drosophila sperm axoneme.

Sdic is a new gene that evolved recently in the lineage of Drosophila melanogaster. It was formed from a duplication and fusion of the gene AnnX, which encodes annexin X, and Cdic, which encodes the intermediate polypeptide chain of the cytoplasmic dynein. The fusion joins AnnX exon 4 with Cdic intron 3, which brings together three putative promoter elements for testes-specific expression of Sdic: the distal conserved element (DCE) and testes-specific element (TSE) are derived from AnnX, and the proximal conserved element (PCE) from Cdic intron 3. Sdic transcription initiates within the PCE, and translation is initiated within the sequence derived from Cdic intron 3, continuing through a 10 base pair insertion that creates a new splice donor site that enables the new coding sequence derived from intron 3 to be joined with the coding sequence of Cdic exon 4. A novel protein is created lacking 100 residues at the amino end that contain sequence motifs essential for the function of cytoplasmic dynein intermediate chains. Instead, the amino end is a hydrophobic region of 16 residues that resembles the amino end of axonemal dynein intermediate chains from other organisms. The downstream portion of Sdic features large deletions eliminating Cdic exons v2 and v3, as well as multiple frameshift deletions or insertions. The new protein becomes incorporated into the tail of the mature sperm and may function as an axonemal dynein intermediate chain. The new Sdic gene is present in about 10 tandem repeats between the wildtype Cdic and AnnX genes located near the base of the X chromosome. The implications of these findings are discussed relative to the origin of new gene functions and the process of speciation.