DEAD box protein DDX1 promotes colorectal tumorigenesis through transcriptional activation of the LGR5 gene.

DDX1, a member of the DEAD box RNA helicase family, plays a critical role in testicular tumors. However, it remains to be clarified whether DDX1 is involved in other types of malignant tumors such as colorectal cancer. We disrupted the DDX1 gene in a human colorectal cancer cell line LoVo using the CRISPR/Cas9-mediated gene-targeting system. DDX1-KO LoVo cells exhibited a much slower growth rate, produced fewer colonies in soft agar medium, and generated smaller solid tumors in nude mice than parental LoVo cells. Such phenotypes of the DDX1-KO cells were mostly reversed by exogenous expression of DDX1. These results indicate that DDX1 is required for tumorigenicity of colorectal cancer cells. In the DDX1-KO cells, the cancer stem cell marker genes LGR5, CD133, ALDH1 and SOX2 were markedly suppressed. Among them, expression of LGR5, which is essential for tumorigenicity of colorectal cancer cells, was restored in the DDX1-transfected DDX1-KO cells. Consistently, the DDX1-KO cells lost sphere-forming capacity in a DDX1-dependent fashion. Reporter and chromatin immunoprecipitation assays revealed that DDX1 directly bound to the -1837 to -1662 region of the enhancer/promoter region of the human LGR5 gene and enhanced its transcription in LoVo cells. Repression of LGR5 by DDX1 knockdown was observed in 2 other human colorectal cancer cell lines, Colo320 and SW837. These results suggest that LGR5 is a critical effector of DDX1 in colorectal cancer cells. The DDX1-LGR5 axis could be a new drug target for this type of malignant cancer.

LIM homeobox transcription factor Lhx2 inhibits skeletal muscle differentiation in part via transcriptional activation of Msx1 and Msx2.

LIM homeobox transcription factor Lhx2 is known to be an important regulator of neuronal development, homeostasis of hair follicle stem cells, and self-renewal of hematopoietic stem cells; however, its function in skeletal muscle development is poorly understood. In this study, we found that overexpression of Lhx2 completely inhibits the myotube-forming capacity of C2C12 cells and primary myoblasts. The muscle dedifferentiation factors Msx1 and Msx2 were strongly induced by the Lhx2 overexpression. Short interfering RNA-mediated knockdown of Lhx2 in the developing limb buds of mouse embryos resulted in a reduction in Msx1 and Msx2 mRNA levels, suggesting that they are downstream target genes of Lhx2. We found two Lhx2 consensus-binding sites in the -2097 to -1189 genomic region of Msx1 and two additional sites in the -536 to +73 genomic region of Msx2. These sequences were shown by luciferase reporter assay to be essential for Lhx2-mediated transcriptional activation. Moreover, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that Lhx2 is present in chromatin DNA complexes bound to the enhancer regions of the Msx1 and Msx2 genes. These data demonstrate that Msx1 and Msx2 are direct transcriptional targets of Lhx2. In addition, overexpression of Lhx2 significantly enhanced the mRNA levels of bone morphogenetic protein 4 and transforming growth factor beta family genes. We propose that Lhx2 is involved in the early stage of skeletal muscle development by inducing multiple differentiation inhibitory factors.

Tumor-suppressive function of protein-tyrosine phosphatase non-receptor type 23 in testicular germ cell tumors is lost upon overexpression of miR142-3p microRNA.

Protein-tyrosine phosphatase non-receptor type 23 (PTPN23) is a candidate tumor suppressor involved in the tumorigenesis of various organs. However, its physiological role(s) and detailed expression profile(s) have not yet been elucidated. We investigated the function and regulation of PTPN23 in the formation of testicular germ cell tumors (TGCTs). Expression of PTPN23 in human TGCT cell lines was significantly lower than that in spermatogonial stem cells in mice. Overexpression of PTPN23 in NEC8, a human TGCT cell line, suppressed soft agar colony formation in vitro and tumor formation in nude mice in vivo. These data indicate that PTPN23 functions as a tumor suppressor in TGCTs. Multiple computational algorithms predicted that the 3' UTR of human PTPN23 is a target for miR-142-3p. A luciferase reporter assay confirmed that miR-142-3p bound directly to the 3' UTR of PTPN23. Introduction of pre-miR-142 in the PTPN23 transfectant of NEC8 led to suppressed expression of PTPN23 and increased soft agar colony formation. Quantitative RT-PCR data revealed a significantly higher expression of miR-142-3p in human seminomas compared with normal testes. No difference in mRNA expression between seminoma and non-seminoma samples was detected by in situ hybridization. Both quantitative RT-PCR and immunohistochemical analyses revealed that PTPN23 expression was significantly lower in TGCTs than in normal testicular tissues. Finally, a lack of PTPN23 protein expression in human TGCTs correlated with a relatively higher miR-142-3p expression. These data suggest that PTPN23 is a tumor suppressor and that repression of PTPN23 expression by miR-142-3p plays an important role in the pathogenesis of TGCTs.

Novel role for the intraflagellar transport protein CMG-1 in regulating the transcription of cyclin-D2, E-cadherin and integrin-alpha family genes in mouse spermatocyte-derived cells.

Capillary morphogenesis gene (CMG)-1 is a mammalian homologue of the intraflagellar transport protein IFT-74/72 of Chlamydomonas. CMG-1 is abundantly expressed in immature stages of male germ-line cells of the adult mouse testis and is required for the expression of cyclin-D2 in GC-2, a mouse premeiotic spermatocyte-derived cell line. In this study, we show that the knockdown of CMG-1 in GC-2 cells leads to down-regulation of E-cadherin, integrin-alpha1, alpha2, alpha10, and alpha11 expression. The ability of the CMG-1-knockdown GC-2 cells to adhere to type-I collagen-coated plates was consequently impaired. Inducible expression of an siRNA-resistant CMG-1 cDNA in these cells rescued the expression of E-cadherin and the integrin-alpha family genes and partially restored adherence to type-I collagen. CMG-1 participates in the transcriptional regulation of cyclin-D2 via a genomic DNA region between -250 and -216 of the mouse cyclin-D2 gene. Closely related sequences were found in the enhancer/promotor regions of E-cadherin and the four integrin-alpha family genes. Based on these data, we propose that CMG-1 serves as a transcriptional regulator of proliferation and adhesion-associated genes in early stage male germ-line cells in the testis.

Disruption of CXC motif chemokine ligand-14 in mice ameliorates obesity-induced insulin resistance.

In obese individuals, white adipose tissue (WAT) is infiltrated by large numbers of macrophages, resulting in enhanced inflammatory responses that contribute to insulin resistance. Here we show that expression of the CXC motif chemokine ligand-14 (CXCL14), which targets tissue macrophages, is elevated in WAT of obese mice fed a high fat diet (HFD) compared with lean mice fed a regular diet. We found that HFD-fed CXCL14-deficient mice have impaired WAT macrophage mobilization and improved insulin responsiveness. Insulin-stimulated phosphorylation of Akt kinase in skeletal muscle was severely attenuated in HFD-fed CXCL14+/- mice but not in HFD-fed CXCL14-/- mice. The insulin-sensitive phenotype of CXCL14-/- mice after HFD feeding was prominent in female mice but not in male mice. HFD-fed CXCL14-/- mice were protected from hyperglycemia, hyperinsulinemia, and hypoadiponectinemia and did not exhibit increased levels of circulating retinol-binding protein-4 and increased expression of interleukin-6 in WAT. Transgenic overexpression of CXCL14 in skeletal muscle restored obesity-induced insulin resistance in CXCL14-/- mice. CXCL14 attenuated insulin-stimulated glucose uptake in cultured myocytes and to a lesser extent in cultured adipocytes. These results demonstrate that CXCL14 is a critical chemoattractant of WAT macrophages and a novel regulator of glucose metabolism that functions mainly in skeletal muscle.

Capillary morphogenesis gene (CMG)-1 is among the genes differentially expressed in mouse male germ line stem cells and embryonic stem cells.

We recently established a technique to expand male germ line stem (GS) cells in long-term culture without losing their spermatogenic capacity. To gain insight into the genetic program of these cells, we compared the mRNA expression profile of GS cells with that of embryonic stem (ES) cells using DNA microarrays. We found 79 genes that were upregulated in GS cells compared to ES cells, including synaptonemal complex protein-1, deleted in azoospermia-like, ubiquitin-conjugating enzyme E2B, and ubiquitin carboxy-terminal hydrolase L1, all of which are functionally important for spermatogenesis. In addition, we identified a cDNA encoding the mouse ortholog of capillary morphogenesis gene (CMG)-1. CMG-1 transcripts were predominantly produced in spermatogonia and spermatocytes in mouse testis. When CMG-1 expression was attenuated in a mouse spermatocyte-derived cell line, GC-2spd(ts), by a target-specific short interfering RNA, the morphology of the cells was changed and the expression of cyclin D2 was abrogated. A reporter assay using a genomic region upstream of the mouse cyclin D2 gene revealed that this downmodulation occurs at the transcriptional level. We detected FLAG-tagged CMG-1 protein in the nuclei of transfected COS7 cells, suggesting that CMG-1 may play a unique role in the transcriptional regulation of the cyclin D2 gene. The upregulated GS genes identified in this study will provide useful information for the future investigation of spermatogonial stem cells and the early phase of male germ cell differentiation.

Spermatogonial cell-mediated activation of an IkappaBzeta-independent nuclear factor-kappaB pathway in Sertoli cells induces transcription of the lipocalin-2 gene.

In spermatogenesis, Sertoli cells serve as supporting cells for the proliferation and differentiation of germ cells. However, it appears that Sertoli cell function is regulated by adjacent spermatogonial cells in the testis because expression of lipocalin-2 mRNA, which encodes an iron-siderophore-binding protein, is barely detectable in Sertoli cells of germ cell-deficient W/Wv mice, and more abundantly expressed in jsd/jsd mice. By employing a coculture system comprising immortalized Sertoli cells (designated as Sertoli-B) and c-Kit(+) spermatogonial cells from 7-d-old mouse testis, we found that lipocalin-2 gene transcription in Sertoli cells is induced by a factor secreted from spermatogonial cells. Transfection of Sertoli-B cells with a series of reporter constructs encompassing an upstream region of the mouse lipocalin-2 gene revealed that a nuclear factor (NF)-kappaB binding consensus sequence in the proximal region of lipocalin-2 gene is responsible for transcriptional activation. A major NF-kappaB component, p65, bound to this region and translocated from the cytoplasm to the nucleus upon stimulation with spermatogonial cell-conditioned medium. Moreover, short interference RNA directed to p65 or a dominant-negative form of IkappaBalpha suppressed the spermatogonial cell factor-mediated transcription of lipocalin-2. However, NF-kappaB-activating inflammatory molecules, such as IL-1beta and lipopolysaccharide, did not induce lipocalin-2 mRNA in Sertoli-B cells and the expression of lipocalin-2 was unaffected in the testis of IkappaBzeta-deficient mice. These results demonstrate that spermatogonial cells regulate lipocalin-2 gene expression in Sertoli cells in a manner distinct from that employed by immune cells.

Runx1 promotes angiogenesis by downregulation of insulin-like growth factor-binding protein-3.

Mouse embryos lacking the Runx1 transcription factor exhibit an angiogenic defect accompanied by the absence of hematopoietic stem cells (HSCs). To ask whether Runx1 plays a direct role in angiogenesis, we established a novel endothelial progenitor cell line, designated AEL-DeltaR1, from the aorta-gonad-mesonephros (AGM) region of Runx1-null mouse. We introduced Runx1 cDNA into AEL-DeltaR1 cells under the doxycycline-inducible promoter. The ability of AEL-DeltaR1 cells to form vascular networks on matrigel was highly enhanced by the restored expression of Runx1. By molecular comparison of mRNAs in AEL-DeltaR1 cells before and after the induction of Runx1, we found that mRNA expression of insulin-like growth factor-binding protein 3 (IGFBP-3) is downregulated by Runx1. Gel retardation and reporter assays revealed that Runx1 binds to the promoter region of mouse IGFBP-3 gene and represses its transcription. When IGFBP-3 was exogenously added in the matrigel assay, the angiogenesis-enhancing activity of Runx1 was suppressed in a dose-dependent manner. These results demonstrate that Runx1 is directly involved in angiogenesis by repression of IGFBP-3 mRNA expression.

Cloning of cDNA encoding a regeneration-associated muscle protease whose expression is attenuated in cell lines derived from Duchenne muscular dystrophy patients.

In the dystrophin-mutant mdx mouse, an animal model for Duchenne muscular dystrophy (DMD), damaged skeletal muscles are efficiently regenerated and thus the animals thrive. The phenotypic differences between DMD patients and the mdx mice suggest the existence of factors that modulate the muscle wasting in the mdx mice. To identify these factors, we searched for mRNAs affected by the mdx mutation by using cDNA microarrays with newly established skeletal muscle cell lines from mdx and normal mice. We found that in the mdx muscle cell line, 12 genes, including L-arginine:glycine amidinotransferase and thymosin beta4, are up-regulated, whereas 7 genes, including selenoprotein P and a novel regeneration-associated muscle protease (RAMP), are down-regulated. Northern blot analysis and in situ hybridization revealed that RAMP mRNA is predominantly expressed in normal skeletal muscle and brain, and its production is enhanced in the regenerating area of injured skeletal muscle in mice. RAMP expression was much lower in individual muscle cell lines derived from biopsies of six DMD patients compared to a normal muscle cell line. These results suggest that RAMP may play a role in the regeneration of skeletal muscle and that its down-regulation could be involved in the progression of DMD in humans.

In vivo differentiation of stem cells in the aorta-gonad-mesonephros region of mouse embryo and adult bone marrow.

OBJECTIVE: Hematopoietic stem cells (HSCs) are thought to be generated from hemangioblasts, the common precursor cells for blood and endothelial cells, in the aorta-gonad-mesonephros (AGM) region of the mouse embryo. The genetic program of HSCs was recently demonstrated to be plastic, but the potential for AGM-region hemangioblasts to be transplanted and to differentiate in vivo has not been well described. Here we examined the fate of donor cells in mice transplanted with CD45(-) AGM cells, which presumably include hemangioblasts. MATERIALS AND METHODS: CD45(-) cells in the AGM region of embryos at 11.5 days post coitum or CD45(+)CD34(-) side population (SP) of cells in adult bone marrow (BM) derived from enhanced green fluorescent protein transgenic mice were transplanted into the liver of busulfan-treated neonatal mice. Two to 6 months after injection of the cells, the contribution of donor-derived cells in the hematopoietic compartment and in various organs was analyzed by flow cytometry and confocal microscopy. RESULTS: CD45(-) cells from the AGM region not only generated peripheral blood cells but also differentiated into endothelial and other nonhematopoietic cells in liver, kidney, lung, small intestine, and uterus in transplanted mice. A similar engrafting pattern was observed in the small intestine of mice transplanted with BM SP/CD45(+) cells, secondary BM-transplanted mice, and lethally irradiated adult mice that received intravenous injections of BM cells. CONCLUSION: A CD45(-) fraction of the AGM region and CD45(+) BM stem cells share the same in vivo potential to differentiate into hematopoietic, endothelial, smooth muscle, and stroma-like cells when transplanted in mice.

Spermatogonia-dependent expression of testicular genes in mice.

Spermatogenesis is initiated by the interaction of germ cells and somatic cells in seminiferous tubules. We used cDNA microarrays and representational difference analysis to identify genes that are expressed in the testis of the jsd/jsd mutant mouse, which contains only type A spermatogonial germ cells and Sertoli cells, but not in the testis of the W/W(v) mutant mouse, where Sertoli cells but few germ cells are present. We isolated 20 known genes and 4 novel genes, including 2 genes encoding lipocalin family members (prostaglandin D synthetase and 24p3) and 2 tumor suppressors (protein tyrosine phosphatase TD14 and Sui1). All 24 of these jsd/jsd-derived genes were highly expressed in the cryptorchid testis as well as in the jsd/jsd testis. This indicates that their selective expression is not directly caused by the as-yet-uncharacterized jsd gene product, but is rather correlated to the cessation of spermatogonial differentiation. In situ hybridization analysis and flow cytometric sorting followed by reverse transcriptase-PCR revealed that these genes are expressed in both the spermatogonial germ cells and the somatic cells in the developing gonads and adult testes. As the mRNAs of these jsd/jsd-derived genes were barely detectable in the W/W(v) testis, we propose that early spermatogonial germ cells regulate the expression of a group of testicular genes.

Novel Inducers and Inhibitors of Differentiation of Friend Erythroleukemia Cells: Application of an Opal Glass Transmission Method to Study of Erythroid Differentiation: (erythroid differentiation/poly(ADP-ribose)/bile acids/opal glass method/Friend leukemia cells).

The potencies of poly(ADP-ribosylation)-inhibitors in inducing erythroid differentiation of Friend erythroleukemia cells were surveyed. Picolinamide and m-aminobenzamide were newly found to be inducers, whereas compounds related caffeine did not induce differentiation. In other series of experiments some bile acids suspected of being tumor promoters were found to inhibit the differentiation like typical tumor promoters such as phorbol esters. These modifications of erythroid differentiation were detected by an opal glass transmission method. This method is simpler than any previously reported methods, and is sufficiently reliable to use in determining hemoglobin in living cells as a quantitative marker of erythroid differentiation.