Julie-Ann Hulin

The novel UDP glycosyltransferase 3A2: cloning, catalytic properties, and tissue distribution.

The human UDP glycosyltransferase (UGT) 3A family is one of three families involved in the metabolism of small lipophilic compounds. Members of these families catalyze the addition of sugar residues to chemicals, which enhances their excretion from the body. The UGT1 and UGT2 family members primarily use UDP glucuronic acid to glucuronidate numerous compounds, such as steroids, bile acids, and therapeutic drugs. We showed recently that UGT3A1, the first member of the UGT3 family to be characterized, is unusual in using UDP N-acetylglucosamine as sugar donor, rather than UDP glucuronic acid or other UDP sugar nucleotides (J Biol Chem 283:36205–36210, 2008). Here, we report the cloning, expression, and characterization of UGT3A2, the second member of the UGT3 family. Like UGT3A1, UGT3A2 is inactive with UDP glucuronic acid as sugar donor. However, in contrast to UGT3A1, UGT3A2 uses both UDP glucose and UDP xylose but not UDP N-acetylglucosamine to glycosidate a broad range of substrates including 4-methylumbelliferone, 1-hydroxypyrene, bioflavones, and estrogens. It has low activity toward bile acids and androgens. UGT3A2 transcripts are found in the thymus, testis, and kidney but are barely detectable in the liver and gastrointestinal tract. The low expression of UGT3A2 in the latter, which are the main organs of drug metabolism, suggests that UGT3A2 has a more selective role in protecting the organs in which it is expressed against toxic insult rather than a more generalized role in drug metabolism. The broad substrate and novel UDP sugar specificity of UGT3A2 would be advantageous for such a function.

Barx2 Is Expressed in Satellite Cells and Is Required for Normal Muscle Growth and Regeneration

Muscle growth and regeneration are regulated through a series of spatiotemporally dependent signaling and transcriptional cascades. Although the transcriptional program controlling myogenesis has been extensively investigated, the full repertoire of transcriptional regulators involved in this process is far from defined. Various homeodomain transcription factors have been shown to play important roles in both muscle development and muscle satellite cell‐dependent repair. Here, we show that the homeodomain factor Barx2 is a new marker for embryonic and adult myoblasts and is required for normal postnatal muscle growth and repair. Barx2 is coexpressed with Pax7, which is the canonical marker of satellite cells, and is upregulated in satellite cells after muscle injury. Mice lacking the Barx2 gene show reduced postnatal muscle growth, muscle atrophy, and defective muscle repair. Moreover, loss of Barx2 delays the expression of genes that control proliferation and differentiation in regenerating muscle. Consistent with the in vivo observations, satellite cell‐derived myoblasts cultured from Barx2−/− mice show decreased proliferation and ability to differentiate relative to those from wild‐type or Barx2+/− mice. Barx2−/− myoblasts show reduced expression of the differentiation‐associated factor myogenin as well as cell adhesion and matrix molecules. Finally, we find that mice lacking both Barx2 and dystrophin gene expression have severe early onset myopathy. Together, these data indicate that Barx2 is an important regulator of muscle growth and repair that acts via the control of satellite cell proliferation and differentiation. STEM CELLS 2012; 30:253–265.

The Homeobox Transcription Factor Barx2 Regulates Plasticity of Young Primary Myofibers

Background Adult mammalian muscle retains incredible plasticity. Muscle growth and repair involves the activation of undifferentiated myogenic precursors called satellite cells. In some circumstances, it has been proposed that existing myofibers may also cleave and produce a pool of proliferative cells that can re-differentiate into new fibers. Such myofiber dedifferentiation has been observed in the salamander blastema where it may occur in parallel with satellite cell activation. Moreover, ectopic expression of the homeodomain transcription factor Msx1 in differentiated C2C12 myotubes has been shown to induce their dedifferentiation. While it remains unclear whether dedifferentiation and redifferentiaton occurs endogenously in mammalian muscle, there is considerable interest in induced dedifferentiation as a possible regenerative tool. Methodology/Principal Findings We previously showed that the homeobox protein Barx2 promotes myoblast differentiation. Here we report that ectopic expression of Barx2 in young immature myotubes derived from cell lines and primary mouse myoblasts, caused cleavage of the syncytium and downregulation of differentiation markers. Microinjection of Barx2 cDNA into immature myotubes derived from primary cells led to cleavage and formation of mononucleated cells that were able to proliferate. However, injection of Barx2 cDNA into mature myotubes did not cause cleavage. Barx2 expression in C2C12 myotubes increased the expression of cyclin D1, which may promote cell cycle re-entry. We also observed differential muscle gene regulation by Barx2 at early and late stages of muscle differentiation which may be due to differential recruitment of transcriptional activator or repressor complexes to muscle specific genes by Barx2. Conclusions/Significance We show that Barx2 regulates plasticity of immature myofibers and might act as a molecular switch controlling cell differentiation and proliferation.

Barx2 and Pax7 Have Antagonistic Functions in Regulation of Wnt Signaling and Satellite Cell Differentiation

The canonical Wnt signaling pathway is critical for myogenesis and can induce muscle progenitors to switch from proliferation to differentiation; how Wnt signals integrate with muscle‐specific regulatory factors in this process is poorly understood. We previously demonstrated that the Barx2 homeobox protein promotes differentiation in cooperation with the muscle regulatory factor (MRF) MyoD. Pax7, another important muscle homeobox factor, represses differentiation. We now identify Barx2, MyoD, and Pax7 as novel components of the Wnt effector complex, providing a new molecular pathway for regulation of muscle progenitor differentiation. Canonical Wnt signaling induces Barx2 expression in muscle progenitors and perturbation of Barx2 leads to misregulation of Wnt target genes. Barx2 activates two endogenous Wnt target promoters as well as the Wnt reporter gene TOPflash, the latter synergistically with MyoD. Moreover, Barx2 interacts with the core Wnt effectors β‐catenin and T cell‐factor 4 (TCF4), is recruited to TCF/lymphoid enhancer factor sites, and promotes recruitment of β‐catenin. In contrast, Pax7 represses the Wnt reporter gene and antagonizes the activating effect of Barx2. Pax7 also binds β‐catenin suggesting that Barx2 and Pax7 may compete for interaction with the core Wnt effector complex. Overall, the data show for the first time that Barx2, Pax7, and MRFs can act as direct transcriptional effectors of Wnt signals in myoblasts and that Barx2 and Wnt signaling participate in a regulatory loop. We propose that antagonism between Barx2 and Pax7 in regulation of Wnt signaling may help mediate the switch from myoblast proliferation to differentiation. Stem Cells 2014;32:1661–1673

Barx2 and Pax7 Regulate Axin2 Expression in Myoblasts by Interaction with β‐Catenin and Chromatin Remodelling

Satellite cells are the resident stem cells of skeletal muscle; quiescent in adults until activated by injury to generate proliferating myoblasts. The canonical Wnt signalling pathway, mediated by T‐cell factor/lymphoid enhancer factor (TCF/LEF) and β‐catenin effector proteins, controls myoblast differentiation in vitro, and recent work suggests that timely termination of the Wnt/β‐catenin signal is important for normal adult myogenesis. We recently identified the Barx2 and Pax7 homeobox proteins as novel components of the Wnt effector complex. Here, we examine molecular and epigenetic mechanisms by which Barx2 and Pax7 regulate the canonical Wnt target gene Axin2, which mediates critical feedback to terminate the transcriptional response to Wnt signals. Barx2 is recruited to the Axin2 gene via TCF/LEF binding sites, recruits β‐catenin and the coactivator GRIP‐1, and induces local H3K‐acetylation. Barx2 also promotes nuclear localization of β‐catenin. Conversely, Pax7 represses Axin2 promoter/intron activity and inhibits Barx2‐mediated H3K‐acetylation via the corepressor HDAC1. Wnt3a not only induces Barx2 mRNA, but also stabilises Barx2 protein in myoblasts; conversely, Wnt3a potently inhibits Pax7 protein expression. As Barx2 promotes myogenic differentiation and Pax7 suppresses it, this novel posttranscriptional regulation of Barx2 and Pax7 by Wnt3a may be involved in the specification of differentiation‐competent and ‐incompetent myoblast populations. Finally, we propose a model for dual function of Barx2 downstream of Wnt signals: activation of myogenic target genes in association with canonical myogenic regulatory factors, and regulation of the negative feedback loop that limits the response of myoblasts to Wnt signals via direct interaction of Barx2 with the TCF/β‐catenin complex. Stem Cells 2016;34:2169–2182

Human dimethylarginine dimethylaminohydrolase 1 inhibition by proton pump inhibitors and the cardiovascular risk marker asymmetric dimethylarginine: in vitro and in vivo significance

Proton pump inhibitor (PPI)-induced inhibition of dimethylarginine dimethylaminohydrolase 1 (DDAH1), with consequent accumulation of the nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA), might explain the increased cardiovascular risk with PPI use. However, uncertainty exists regarding whether clinical PPI concentrations significantly inhibit DDAH1 under linear initial rate conditions, and whether PPI-induced DDAH1 inhibition significantly increases ADMA in humans. DDAH1 inhibition by esomeprazole, omeprazole, pantoprazole, lansoprazole and rabeprazole was determined by quantifying DDAH1-mediated L-citrulline formation in vitro. Plasma ADMA was measured in PPI users (n = 134) and non-users (n = 489) in the Hunter Community Study (HCS). At clinical PPI concentrations (0.1–10 μmol/L), DDAH1 retained >80% activity vs. baseline. A significant, reversible, time-dependent inhibition was observed with lansoprazole (66% activity at 240 min, P = 0.034) and rabeprazole (25% activity at 240 min, P < 0.001). In regression analysis, PPI use was not associated with ADMA in HCS participants (beta 0.012, 95% CI −0.001 to 0.025, P = 0.077). Furthermore, there were no differences in ADMA between specific PPIs (P = 0.748). At clinical concentrations, PPIs are weak, reversible, DDAH1 inhibitors in vitro. The lack of significant associations between PPIs and ADMA in HCS participants questions the significance of DDAH1 inhibition as a mechanism explaining the increased cardiovascular risk reported with PPI use.

MiR-193b regulates breast cancer cell migration and vasculogenic mimicry by targeting dimethylarginine dimethylaminohydrolase 1

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is responsible for metabolism of an endogenous inhibitor of nitric oxide synthase (NOS), asymmetric dimethylarginine (ADMA), which plays a key role in modulating angiogenesis. In addition to angiogenesis, tumours can establish a vascular network by forming vessel-like structures from tumour cells; a process termed vasculogenic mimicry (VM). Here, we identified over-expression of DDAH1 in aggressive MDA-MB-231, MDA-MB-453 and BT549 breast cancer cell lines when compared to normal mammary epithelial cells. DDAH1 expression was inversely correlated with the microRNA miR-193b. In DDAH1+ MDA-MB-231 cells, ectopic expression of miR-193b reduced DDAH1 expression and the conversion of ADMA to citrulline. In DDAH1− MCF7 cells, inhibition of miR-193b elevated DDAH1 expression. Luciferase reporter assays demonstrated DDAH1 as a direct target of miR-193b. MDA-MB-231 cells organised into tube structures in an in vitro assay of VM, which was significantly inhibited by DDAH1 knockdown or miR-193b expression. Mechanistically, we found miR-193b regulates cell proliferation and migration of MDA-MB-231 cells, whilst DDAH1 knockdown inhibited cell migration. These studies represent the first evidence for DDAH1 expression, regulation and function in breast cancer cells, and highlights that targeting DDAH1 expression and/or enzymatic activity may be a valid option in the treatment of aggressive breast cancers.

Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2-8) in Normal and Cancerous Breast and Prostate Cells.

Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2–8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer (PCGEM1, PEG3, EPHA3, and EFNB2) or other types of human cancers (TOX3, ST8SIA4, and SLITRK3), and genes that are diagnostic/prognostic biomarkers of prostate cancer (GRINA3, and BCHE).

Cooperative Regulation of Intestinal UDP-Glucuronosyltransferases 1A8, -1A9, and 1A10 by CDX2 and HNF4α Is Mediated by a Novel Composite Regulatory Element

The gastrointestinal tract expresses several UDP-glucuronosyltransferases (UGTs) that act as a first line of defense against dietary toxins and contribute to the metabolism of orally administered drugs. The expression of UGT1A8, UGT1A9, and UGT1A10 in gastrointestinal tissues is known to be at least partly directed by the caudal homeodomain transcription factor, CDX2. We sought to further define the factors involved in regulation of the UGT1A8-1A10 genes and identified a novel composite element located within the proximal promoters of these three genes that binds to both CDX2 and the hepatocyte nuclear factor (HNF) 4α, and mediates synergistic activation by these factors. We also show that HNF4α and CDX2 are required for the expression of these UGT genes in colon cancer cell lines, and show robust correlation of UGT expression with CDX2 and HNF4α levels in normal human colon. Finally, we show that these factors are involved in the differential expression pattern of UGT1A8 and UGT1A10, which are intestinal specific, and that of UGT1A9, which is expressed in both intestine and liver. These studies lead to a model for the developmental patterning of UGT1A8, UGT1A9, and UGT1A10 in hepatic and/or extrahepatic tissues involving discrete regulatory modules that may function (independently and cooperatively) in a context-dependent manner.

Intergenic splicing between four adjacent UGT genes (2B15, 2B29P2, 2B17, 2B29P1) gives rise to variant UGT proteins that inhibit glucuronidation via protein-protein interactions

Recent studies have investigated alternative splicing profiles of UDP-glucuronosyltransferase (UGT) genes and identified over 130 different alternatively spliced UGT transcripts. Although UGT genes are highly clustered, the formation of chimeric transcripts by intergenic splicing between two or more UGT genes has not yet been reported. This study identified 12 chimeric transcripts (chimeras A–L) containing exons from two or three genes of the four neighboring UGT genes (UGT2B15, UGT2B29P2, UGT2B17, and UGT2B29P1) in human liver and prostate cancer cells. These chimeras typically contain the first five exons of UGT2B15 or UGT2B17 (exons 1–5) spliced to a terminal exon (exon 6) from a downstream UGT gene. Hence they encode truncated UGTs with novel C-terminal peptides. Functional assays of representative chimeric UGT proteins (termed chimeric UGT2B15 and chimeric UGT2B17) showed that they are inactive and can repress the activity of wild-type UGTs. Coimmunoprecipitation assays demonstrated heterotypic interactions between chimeric UGT2B15 (or chimeric UGT2B17) and the UGT2B7 protein. Thus oligomerization of the chimeric UGTs with wild-type UGTs may explain their inhibitory activity. Studies in breast and prostate cancer cells showed that both wild-type and chimeric UGT2B15 and UGT2B17 transcripts are regulated in a similar way at the transcriptional level by sex hormones through their canonical promoters but are differentially regulated at the post-transcriptional level by micro-RNA 376c via their unique 3′-untranslated regions. In conclusion, the formation of chimeric transcripts by intergenic splicing among UGT genes represents a novel mechanism contributing to the diversity of the human UGT transcriptome and proteome. The differential post-transcriptional regulation of wild-type and variant transcripts by micro-RNAs may contribute to their deregulated expression in cancer.