PLAG1 deficiency impairs spermatogenesis and sperm motility in mice.

Deficiency in pleomorphic adenoma gene 1 (PLAG1) leads to reduced fertility in male mice, but the mechanism by which PLAG1 contributes to reproduction is unknown. To investigate the involvement of PLAG1 in testicular function, we determined (i) the spatial distribution of PLAG1 in the testis using X-gal staining; (ii) transcriptomic consequences of PLAG1 deficiency in knock-out and heterozygous mice compared to wild-type mice using RNA-seq; and (iii) morphological and functional consequences of PLAG1 deficiency by determining testicular histology, daily sperm production and sperm motility in knock-out and wild-type mice. PLAG1 was sparsely expressed in germ cells and in Sertoli cells. Genes known to be involved in spermatogenesis were downregulated in the testes of knock-out mice, as well as Hsd17b3, which encodes a key enzyme in androgen biosynthesis. In the absence of Plag1, a number of genes involved in immune processes and epididymis-specific genes were upregulated in the testes. Finally, loss of PLAG1 resulted in significantly lowered daily sperm production, in reduced sperm motility, and in several animals, in sloughing of the germinal epithelium. Our results demonstrate that the subfertility seen in male PLAG1-deficient mice is, at least in part, the result of significantly reduced sperm output and sperm motility.

Synthesis and Characterization of Curcumin-Functionalized HP-ß-CD-Modified GoldMag Nanoparticles as Drug Delivery Agents.

Curcumin, a polyphenol extracted from turmeric (Curcuma longa), has emerged as a potent multimodal cancer-preventing agent. It may attenuate the spread of cancer and render chemotherapy more effective. However, curcumin is neither well absorbed nor well retained in the blood, resulting in low efficacy. In an attempt to enhance the potency and to improve the bioavailability of curcumin, new delivery agents, hydroxypropyl-beta-cyclodextrin (HP-ß-CD)-modified GoldMag nanoparticles (CD-GMNs) were designed and synthesized to incorporate curcumin. The CD-GMNs were characterized by Fourier Transform Infrared Spectroscopy (FT-IR), Thermo-gravimetric Analysis (TGA), X-ray Diffraction (XRD), Dynamic Light Scattering measurements (DLS), Transmission Electron Microscopy (TEM) and Vibrating Sample Magnetometer (VSM) analyses. For the magnetic carrier of CD-GMNs, the content of HP-ß-CD was 26.9 wt%. CD-GMNs have a saturation magnetization of 22.7 emu/g with an average hydrodynamic diameter of 80 nm. The curcumin loading, encapsulation efficiency and releasing properties in vitro were also investigated. The results showed that the drug encapsulation ratio was 88% and the maximum curcumin loading capacity of CD-GMNs was 660 µg/5 mg. In vitro drug release studies showed a controlled and pH-sensitive curcumin release over a period of one week. Collectively, our data suggest that HP-ß-CD-modified GoldMag nanoparticles can be considered to form a promising delivery system for curcumin to tumor sites. Targeting can be achieved by the combined effects of the application of an external magnetic field and the effect on drug release of lower pH values often found in the tumor microenvironment.

Emerging role of PLAG1 as a regulator of growth and reproduction.

Pleomorphic adenoma gene 1 (PLAG1) belongs to the PLAG family of zinc finger transcription factors along with PLAG-like 1 and PLAG-like 2. The PLAG1 gene is best known as an oncogene associated with certain types of cancer, most notably pleomorphic adenomas of the salivary gland. While the mechanisms of PLAG1-induced tumorigenesis are reasonably well understood, the role of PLAG1 in normal physiology is less clear. It is known that PLAG1 is involved in cell proliferation by directly regulating a wide array of target genes, including a number of growth factors such as insulin-like growth factor 2. This is likely to be a central mode of action for PLAG1 both in embryonic development and in cancer. The phenotype of Plag1 knockout mice suggests an important role for PLAG1 also in postnatal growth and reproduction, as PLAG1 deficiency causes growth retardation and reduced fertility. A role for PLAG1 in growth and reproduction is further corroborated by genome-wide association studies in humans and domestic animals in which polymorphisms in the PLAG1 genomic region are associated with body growth and reproductive traits. Here we review the current evidence for PLAG1 as a regulator of growth and fertility and discuss possible endocrine mechanisms involved.

Curcumin affects proprotein convertase activity: elucidation of the molecular and subcellular mechanism.

Proprotein convertases (PCs) form a group of serine endoproteases that are essential for the activation of proproteins into their active form. Some PCs have been proposed to be potential therapeutic targets for cancer intervention because elevated PC activity has been observed in many different cancer types and because many of the PC substrates, such as pro-IGF-1R, pro-TGF-beta, pro-VEGF, are involved in signaling pathways related to tumor development. Curcumin, reported to possess anticancer activity, also affects many of these pathways. We therefore investigated the effect of curcumin on PC activity. Our results show that curcumin inhibits PC activity in a cell lysate-based assay but not in vitro. PC zymogen maturation in the endoplasmic reticulum appears to be inhibited by curcumin. Treating cells with thapsigargin or cyclopiazonic acid, two structurally unrelated inhibitors of the sarco- and endoplasmic reticulum Ca(2+)ATPase (SERCA), also hampered both the PC zymogen maturation and the PC activity. Importantly, curcumin, like the SERCA inhibitors, impaired ATP-driven (45)Ca(2+) uptake in the endoplasmic reticulum. These results indicate that curcumin likely restrains PC activity by inhibiting SERCA-mediated Ca(2+)-uptake activity. Experiments in three colon cancer cell lines confirm that curcumin inhibits both the (45)Ca(2+) uptake and PC activity, notably the processing of pro-IGF-1R. Both curcumin and thapsigargin inhibit the anchorage-independent growth of these three colon carcinoma cell lines. In conclusion, our findings indicate that curcumin inhibits PC zymogen maturation and consequently PC activity and that its inhibitory effect on Ca(2+) uptake into the ER allows and is sufficient to explain this phenomenon.

A novel magnetic nanoparticle drug carrier for enhanced cancer chemotherapy.

BACKGROUND: Magnetic nanoparticles (NPs) loaded with antitumor drugs in combination with an external magnetic field (EMF)-guided delivery can improve the efficacy of treatment and may decrease serious side effects. The purpose of this study was 1) to investigate application of PEG modified GMNPs (PGMNPs) as a drug carrier of the chemotherapy compound doxorubicin (DOX) in vitro; 2) to evaluate the therapeutic efficiency of DOX-conjugated PGMNPs (DOX-PGMNPs) using an EMF-guided delivery in vivo. METHODS: First, DOX-PGMNPs were synthesized and the cytotoxicity of DOX-PGMNPs was assessed in vitro. Second, upon intravenous administration of DOX-PMGPNs to H22 hepatoma cell tumor-bearing mice, the DOX biodistribution in different organs (tissues) was measured. The antitumor activity was evaluated using different treatment strategies such as DOX-PMGPNs or DOX-PMGPNs with an EMF-guided delivery (DOX-PGMNPs-M). RESULTS: The relative tumor volumes in DOX-PGMNPs-M, DOX-PGMNPs, and DOX groups were 5.46±1.48, 9.22±1.51, and 14.8±1.64, respectively (each p<0.05), following treatment for 33 days. The life span of tumor-bearing mice treated with DOX-PGMNPs-M, DOX-PGMNPs, and DOX were 74.8±9.95, 66.1±13.5, and 31.3±3.31 days, respectively (each p<0.05). CONCLUSION: This simple and adaptive nanoparticle design may accommodate chemotherapy for drug delivery optimization and in vivo drug-target definition in system biology profiling, increasing the margin of safety in treatment of cancers in the near future.

Generation and characterization of non-competitive furin-inhibiting nanobodies.

The PC (proprotein convertase) furin cleaves a large variety of proproteins and hence plays a major role in many pathologies. Therefore furin inhibition might be a good strategy for therapeutic intervention, and several furin inhibitors have been generated, although none are entirely furin-specific. To reduce potential side effects caused by cross-reactivity with other proteases, dromedary heavy-chain-derived nanobodies against catalytically active furin were developed as specific furin inhibitors. The nanobodies bound only to furin but not to other PCs. Upon overexpression in cell lines, they inhibited the cleavage of two different furin substrates, TGFß (transforming growth factor ß) and GPC3 (glypican 3). Purified nanobodies could inhibit the cleavage of diphtheria toxin into its enzymatically active A fragment, but did not inhibit cleavage of a small synthetic peptide-based substrate, suggesting a mode-of-action based on steric hindrance. The dissociation constant of purified nanobody 14 is in the nanomolar range. The nanobodies were non-competitive inhibitors with an inhibitory constant in the micromolar range as demonstrated by Dixon plot. Furthermore, anti-furin nanobodies could protect HEK (human embryonic kidney)-293T cells from diphtheria-toxin-induced cytotoxicity as efficiently as the PC inhibitor nona-D-arginine. In conclusion, these antibody-based single-domain nanobodies represent the first generation of highly specific non-competitive furin inhibitors.

Pdx1- and Ngn3-Cre-mediated PLAG1 expression in the pancreas leads to endocrine hormone imbalances that affect glucose metabolism.

Pleomorphic adenoma gene-like 1 (PLAGL1) has been linked to transient neonatal diabetes mellitus. Here, we investigated the role of the related pleomorphic adenoma gene 1 (PLAG1) in glucose homeostasis. PLAG1 transgenic mice in which expression of the PLAG1 transgene can be targeted to different organs by Cre-mediated modulation were crossed with Pdx1-Cre or Ngn3-Cre mice, resulting in double transgenic P1-Pdx1Cre or P1-Ngn3Cre mice, respectively. P1-Pdx1Cre and P1-Ngn3Cre mice developed hyperplasia of pancreatic islets due to increased ß- and δ- but not α-cell proliferation. In young P1-Pdx1Cre mice (less than 15 weeks) there was a balanced increase in the pancreatic content of insulin and somatostatin, which was associated with normoglycemia. In older P1-Pdx1Cre mice the pancreatic somatostatin content far exceeded that of insulin, leading to the progressive development of severe hypoglycemia beyond 30 weeks. In contrast, in older P1-Ngn3Cre mice the relative increase of the pancreatic insulin content exceeded that of somatostatin and these mice remained normoglycemic. In conclusion, forced expression of PLAG1 under the control of the Pdx1 or Ngn3 promoter in murine pancreas induces different degrees of endocrine hormone imbalances within the pancreas, which is associated with hypoglycemia in P1-Pdx1Cre mice but not P1-Ngn3Cre mice. These results suggest that once stem cell-derived islet transplantations become possible, the appropriate balance between different hormone-producing cells will need to be preserved to prevent deregulated glucose metabolism.

Increased beta-cell mass by islet transplantation and PLAG1 overexpression causes hyperinsulinemic normoglycemia and hepatic insulin resistance in mice.

OBJECTIVE: It is believed that an organism remains normoglycemic despite an increase in the beta-cell mass because of decreased insulin production by beta-cells on a per-cell basis. However, some transgenic mouse models with beta-cell hyperplasia suggest that insulin production remains excessive and that normoglycemia is maintained by insulin resistance. METHODS: Here, we investigated the effect of an increased beta-cell mass on glycemia and insulin resistance by grafting excess normal islets in normoglycemic mice, as well as using targeted PLAG1 expression in beta-cells, which leads to beta-cell expansion. RESULTS: In both models, fasting plasma insulin levels were increased, even though animals were normoglycemic. After an intraperitoneal glucose tolerance test, plasma insulin levels increased, which was associated with improved glucose clearing. Under these conditions, normoglycemia is maintained by hepatic insulin resistance as demonstrated by hyperinsulinemic euglycemic clamp experiments. CONCLUSIONS: In conclusion, we demonstrate that when excess beta-cells are grafted, insulin production on a per beta-cell basis is not sufficiently decreased, leading to hyperinsulinemia and hepatic insulin resistance. This observation might be important for the design of stem cell-based islet replacement therapies.

SCAMP5, NBEA and AMISYN: three candidate genes for autism involved in secretion of large dense-core vesicles.

Autism is a neurodevelopmental disorder characterized by impaired social reciprocity, impaired communication and stereotypical behaviors. Despite strong evidence for a genetic basis, few susceptibility genes have been identified. Here, we describe the positional cloning of SCAMP5, CLIC4 and PPCDC as candidate genes for autism, starting from a person with idiopathic, sporadic autism carrying a de novo chromosomal translocation. One of these genes, SCAMP5 is silenced on the derivative chromosome, and encodes a brain-enriched protein involved in membrane trafficking, similar to the previously identified candidate genes NBEA and AMISYN. Gene silencing of Nbea, Amisyn and Scamp5 in mouse beta-TC3 cells resulted in a 2-fold increase in stimulated secretion of large dense-core vesicles (LDCVs), while overexpression suppressed secretion. Moreover, ultrastructural analysis of blood platelets from the patients with haploinsufficieny of one of the three candidate genes, showed morphological abnormalities of dense-core granules, which closely resemble LDCVs. Taken together, this study shows that in three independent patients with autism three different negative regulators of LDCV secretion are affected, respectively, suggesting that in at least a subgroup of patients the regulation of neuronal vesicle trafficking may be involved in the pathogenesis of autism.

Targeted disruption of the mouse Lipoma Preferred Partner gene.

LPP (Lipoma Preferred Partner) is a zyxin-related cell adhesion protein that is involved in the regulation of cell migration. We generated mice with a targeted disruption of the Lpp gene and analysed the importance of Lpp for embryonic development and adult functions. Aberrant Mendelian inheritance in heterozygous crosses suggested partial embryonic lethality of Lpp(-/-) females. Fertility of Lpp(-/-) males was proven to be normal, however, females from Lpp(-/-) x Lpp(-/-) crosses produced a strongly reduced number of offspring, probably due to a combination of female embryonic lethality and aberrant pregnancies. Apart from these developmental and reproductive abnormalities, Lpp(-/-) mice that were born reached adulthood without displaying any additional macroscopic defects. On the other hand, Lpp(-/-) mouse embryonic fibroblasts exhibited reduced migration capacity, reduced viability, and reduced expression of some Lpp interaction partners. Finally, we discovered a short nuclear form of Lpp, expressed mainly in testis via an alternative promoter.

The human pseudoxanthoma elasticum gene ABCC6 is transcriptionally regulated by PLAG family transcription factors.

Mutations in the ABCC6 gene are known as causative factors of pseudoxanthoma elasticum (PXE), a connective tissue calcification disorder, but the molecular mechanism of pathogenesis or the physiological function of ABCC6 protein is the subject of intense debate. The ABCC6 gene expression is tightly regulated at the transcriptional level and its tissue-specific distribution is consistent with PXE being a metabolic disease caused by failure of ABCC6 function in organs distant from the diseased sites. In an effort to provide clues to its role by elucidating the mechanisms of its regulation, we identified ABCC6 as a target gene for transcriptional induction by PLAG1 and PLAGL1, transcription factors from the PLAG family of cell cycle progression-related DNA-binding proteins. Both these factors are shown to bind to the same single consensus-binding element in the ABCC6 proximal promoter in cell lines of hepatic and renal origin by reporter gene assay, electrophoretic mobility shift assay and chromatin immunoprecipitation. PLAG-mediated ABCC6 transactivation may play an important role in determining the level of tissue-specific expression of this gene. The described mechanism can also find potential application in therapeutic interventions in forms of PXE related to impaired ABCC6 expression.

Role of furin in granular acidification in the endocrine pancreas: identification of the V-ATPase subunit Ac45 as a candidate substrate.

Furin is a proprotein convertase which activates a variety of regulatory proteins in the constitutive exocytic and endocytic pathway. The effect of genetic ablation of fur was studied in the endocrine pancreas to define its physiological function in the regulated secretory pathway. Pdx1-Cre/loxP furin KO mice show decreased secretion of insulin and impaired processing of known PC2 substrates like proPC2 and proinsulin II. Both secretion and PC2 activity depend on granule acidification, which was demonstrated to be significantly decreased in furin-deficient beta cells by using the acidotrophic agent 3-(2,4-dinitroanilino)-3'amino-N-methyldipropylamine (DAMP). Ac45, an accessory subunit of the proton pump V-ATPase, was investigated as a candidate substrate. Ac45 is highly expressed in islets of Langerhans and furin was able to cleave Ac45 ex vivo. Furthermore, the exact cleavage site was determined. In addition, reduced regulated secretion and proinsulin II processing could be obtained in the insulinoma cell line betaTC3 by downregulation of either furin or Ac45. Together, these data establish an important role for furin in regulated secretion, particularly in intragranular acidification most likely due to impaired processing of Ac45.

Adenomyoepitheliomatous lesions of the mammary glands in transgenic mice with targeted PLAG1 overexpression.

PLAG1 proto-oncogene overexpression has been causally linked to multiple tumors, highlighting its broad tumorigenic relevance. Here, the oncogenic potential of PLAG1 in mammary gland tumorigenesis was investigated in PLAG1 transgenic mice. To target mammary glands, mice of 2 independent PLAG1 transgenic strains, PTMS1 and PTMS2, in which PLAG1 expression can be modulated by Cre-mediation, were crossed with MMTV-Cre transgenic mice, resulting in P1-MCre and P2-MCre offspring, respectively. Hundred percentage of P1-MCre female mice showed mammary gland hyperplasia, caused by adenomyoepithelial adenosis, at 8 weeks. The tumorigenic process could not be studied further in P1-MCre mice, because concomitant fast-growing salivary gland tumors required euthanasia. Sixteen percentage of P2-MCre females developed mammary gland adenomyoepitheliomas within 30-45 weeks, and none displayed concomitant salivary gland tumors. To further study mammary gland tumorigenesis in PTMS1-derived mice, intercrossing with WAP-Cre transgenic mice, resulting in P1-WAPCre mice, was performed to target PLAG1 expression more specifically to mammary glands. Eighty percentage of such mice developed adenomyoepitheliomas within 53-88 weeks. All PLAG1-induced adenomyoepitheliomas revealed expression upregulation of Igf2/H19, Dlk1/Gtl2, Igfbps and Wnt signaling genes (Wnt6, Cyclin D1). Collectively, these results establish the oncogenic potential of PLAG1 in mammary glands of mice and point towards contributing roles of Igf and Wnt signaling.

Lpp is involved in Wnt/PCP signaling and acts together with Scrib to mediate convergence and extension movements during zebrafish gastrulation.

The zyxin-related LPP protein is localized at focal adhesions and cell-cell contacts and is involved in the regulation of smooth muscle cell migration. A known interaction partner of LPP in human is the tumor suppressor protein SCRIB. Knocking down scrib expression during zebrafish embryonic development results in defects of convergence and extension (C&E) movements, which occur during gastrulation and mediate elongation of the anterior-posterior body axis. Mediolateral cell polarization underlying C&E is regulated by a noncanonical Wnt signaling pathway constituting the vertebrate planar cell polarity (PCP) pathway. Here, we investigated the role of Lpp during early zebrafish development. We show that morpholino knockdown of lpp results in defects of C&E, phenocopying noncanonical Wnt signaling mutants. Time-lapse analysis associates the defective dorsal convergence movements with a reduced ability to migrate along straight paths. In addition, expression of Lpp is significantly reduced in Wnt11 morphants and in embryos overexpressing Wnt11 or a dominant-negative form of Rho kinase 2, which is a downstream effector of Wnt11, suggesting that Lpp expression is dependent on noncanonical Wnt signaling. Finally, we demonstrate that Lpp interacts with the PCP protein Scrib in zebrafish, and that Lpp and Scrib cooperate for the mediation of C&E.

Upregulation of Igf and Wnt signalling associated genes in pleomorphic adenomas of the salivary glands in PLAG1 transgenic mice.

The Pleomorphic adenoma gene 1 (PLAG1) is involved in various human neoplasias, including pleomorphic adenomas of the salivary glands. Moreover, the oncogenic role of PLAG1 was clearly demonstrated in two independent PLAG1 transgenic mouse founders, in which PLAG1 expression could be targeted to different tissues using the Cre/loxP system. MMTV-Cre-mediated targeted overexpression of PLAG1 in the salivary glands of double transgenic offspring mice, referred to as P1-MCre and P2-MCre mice, induced pleomorphic adenomas in this organ. Igf2, a genuine PLAG1 target gene, was highly upregulated in those tumours as well as in human pleomorphic adenomas of the salivary glands. These and previous observations in other PLAG1-induced tumours e.g. breast adenomyoepitheliomas emphasize the importance of Igf upregulation in such tumours. In this study, further evidence for the role of Igf2 in PLAG1-induced tumourigenesis, is reported. Inactivation of Igf2 in P1-MCre mice leads to a significant delay in tumour development. Since tumour development is not fully abrogated by inactivation of Igf2, other signalling pathways are likely to contribute to PLAG1-induced tumourigenesis as well. Further studies revealed that several genes such as H19, Dlk1, Gtl2, Igfbp2, Igfbp3 and genes involved in Wnt signalling, such as Wnt6, Cyclin D1 and beta-catenin are upregulated in P1-MCre mice in which Igf2 is inactivated. In conclusion, we clearly demonstrate upregulation of several genes associated with Igf and Wnt signalling in PLAG1-induced pleomorphic adenomas. Furthermore, inactivation of Igf2 does not affect upregulation of genes associated with Wnt signalling, which might suggest that both signalling pathways are involved.

The HMGA proteins: a myriad of functions (Review).

The 'high mobility group' HMGA protein family consists of four members: HMGA1a, HMGA1b and HMGA1c, which result from translation of alternative spliced forms of one gene and HMGA2, which is encoded for by another gene. HMGA proteins are characterized by three DNA-binding domains, called AT-hooks, and an acidic carboxy-terminal tail. HMGA proteins are architectural transcription factors that both positively and negatively regulate the transcription of a variety of genes. They do not display direct transcriptional activation capacity, but regulate gene expression by changing the DNA conformation by binding to AT-rich regions in the DNA and/or direct interaction with several transcription factors. In this way, they influence a diverse array of normal biological processes including cell growth, proliferation, differentiation and death. Both HMGA1 and HMGA2 are hardly detectable in normal adult tissue but are abundantly and ubiquitously expressed during embryonic development. In malignant epithelial tumors as well as in leukemia, however, expression of HMGA1 is again strongly elevated to embryonic levels thus leading to ectopic expression of (fetal) target genes. HMGA2 overexpression also has a causal role in inducing neoplasia. Besides overexpression of full length HMGA proteins in different tumors, the HMGA genes are often involved in chromosomal rearrangements. Such translocations are mostly detected in benign tumors of mesenchymal origin and are believed to be one of the most common chromosomal rearrangements in human neoplasia. To provide clarity in the abundance of articles on this topic, this review gives a general overview of the nuclear functions and regulation of the HMGA genes and corresponding proteins.

aP2-Cre-mediated expression activation of an oncogenic PLAG1 transgene results in cavernous angiomatosis in mice.

The developmentally regulated PLAG1 proto-oncogene has been implicated in the development of various human tumor types, such as pleomorphic salivary gland adenomas, lipoblastomas, hepatoblastomas and AML. In previous studies, we generated two independent PLAG1 transgenic founder strains, PTMS1 and PTMS2, in which PLAG1 could be activated via Cre-mediated excision of a stop cassette. With these founders, PLAG1-induced tumor formation in salivary and mammary glands of mice was studied. To further delineate the oncogenic spectrum of PLAG1 in mice, we induced aP2-Cre-mediated overexpression of PLAG1 in offspring from crossbreeding PTMS1 mice with aP2-Cre transgenic mice. More than 80% of aP2-Cre(+/-)/PLAG1(+/-) (P1-ACre) mice developed a vascular tumor type within one year, which could be classified histopathologically as cavernous angiomatosis. The lesions occurred in various regions of the mouse body but almost exclusively in the immediate surrounding of fat cells. Validation of available PLAG1-induced gene expression profiling data, using targeted tissues, revealed that expression activation of PLAG1 is functional because it leads to elevated levels of PLAG1 target gene transcripts in those tissues, such as for instance those of H19, Dlk1, and Igf-2, similarly as observed in PLAG1-induced salivary and mammary gland tumors. In conclusion, we present the first evidence that links PLAG1 to the molecular pathogenesis of vascular tumorigenesis, known as cavernous angiomatosis, with the possible involvement of Igf signaling and, moreover, further delineate the oncogenic spectrum of PLAG1 in mice, increasing the potential of this transgenic mouse tumor model system for research and therapeutic drug testing.

Loss of the PlagL2 transcription factor affects lacteal uptake of chylomicrons.

Enterocytes assemble dietary lipids into chylomicron particles that are taken up by intestinal lacteal vessels and peripheral tissues. Although chylomicrons are known to assemble in part within membrane secretory pathways, the modifications required for efficient vascular uptake are unknown. Here we report that the transcription factor pleomorphic adenoma gene-like 2 (PlagL2) is essential for this aspect of dietary lipid metabolism. PlagL2(-/-) mice die from postnatal wasting owing to failure of fat absorption. Lipids modified in the absence of PlagL2 exit from enterocytes but fail to enter interstitial lacteal vessels. Dysregulation of enterocyte genes closely linked to intracellular membrane transport identified candidate regulators of critical steps in chylomicron assembly. PlagL2 thus regulates important aspects of dietary lipid absorption, and the PlagL2(-/-) animal model has implications for the amelioration of obesity and the metabolic syndrome.

HMGA2 regulates transcription of the Imp2 gene via an intronic regulatory element in cooperation with nuclear factor-kappaB.

IMP2 (insulin-like growth factor-II mRNA binding protein 2) is an oncofetal protein that is aberrantly expressed in several types of cancer. We recently identified the Imp2 gene as a target gene of the architectural transcription factor HMGA2 (high mobility group A2) and its tumor-specific truncated form HMGA2Tr. In this study, we investigated the mechanism via which HMGA2 regulates Imp2 gene expression. We show that HMGA2 and HMGA2Tr directly regulate transcription of the Imp2 gene by binding to an AT-rich regulatory region located in the first intron. In reporter experiments, we show that this AT-rich regulatory region mimics the response of the endogenous Imp2 gene to HMGA2 and HMGA2Tr. Furthermore, we show that a consensus nuclear factor-kappaB (NF-kappaB) binding site located immediately adjacent to the AT-rich regulatory region binds NF-kappaB and that NF-kappaB and HMGA2 cooperate to regulate Imp2 gene expression. Finally, we provide evidence that there is a strong and statistically significant correlation between HMGA2 and IMP2 gene expression in human liposarcomas.

PLAG1, the prototype of the PLAG gene family: versatility in tumour development (review).

Recent studies of human tumours as well as genetically engineered mouse tumour models have established the importance and versatility of the PLAG1 oncogene in tumourigenesis. The PLAG1 proto-oncogene was discovered by studying the t(3;8)(p21;q12) chromosome translocation, which frequently occurs in human pleomorphic adenomas of the salivary glands. PLAG1 encodes a developmentally regulated, SUMOylated and phosphorylated zinc finger transcription factor, recognizes a specific bipartite DNA consensus sequence regulating expression of a spectrum of target genes, and has two structurally related family members, i.e. the PLAGL1 and PLAGL2 gene. Ectopic PLAG1 overexpression, in many cases due to promoter swapping, causes deregulation of expression of a variety of PLAG1 target genes. This was established by microarray analysis, which indicated that the oncogenic capability of PLAG1 is mediated, at least partly, by the IGF-II mitogenic signaling pathway. Oncogenic activation of PLAG1 is also a crucial event in other human tumours, including lipoblastoma, hepatoblastoma, and AML. The oncogenic potential of PLAG1 has been confirmed in in vitro experiments, which also established IGF-II and IGF-IR as key pathway elements, similarly as in many human tumours. Furthermore, generation of conditional PLAG1 transgenic mouse strains revealed tumour development in a variety of targeted tissues, establishing the versatility of the PLAG1 oncogene and pointing towards a window of opportunity for therapeutic intervention studies. In contrast to the pleiotropic oncogenic potential of PLAG1, its family member PLAGL1, which is localized in an imprinted region on chromosome 6q24-25, is defined by various studies as a tumour-suppressor gene. Finally, the PLAGL2 family member is not only structurally but also functionally more closely related to PLAG1 and has recently also been implicated in AML, both in humans and in genetically modified mice. Collectively, these observations emphasize a more general importance of the PLAG1 gene in tumour development. In light of the fact that IGF-IR is implicated in many human tumours, the diversity in PLAG1-induced mouse tumour models, most of which seem to involve Igf2 signaling, provides useful in vivo platforms to start testing the effects of inhibitors, such as Igf-1r inhibitors, on tumour development in distinct tissues or organ types.