JMJD5, a Jumonji C (JmjC) domain-containing protein, negatively regulates osteoclastogenesis by facilitating NFATc1 protein degradation.

Osteoclastogenesis is a highly regulated process governed by diverse classes of regulators. Among them, nuclear factor of activated T-cells calcineurin-dependent 1 (NFATc1) is the primary osteoclastogenic transcription factor, and its expression is transcriptionally induced during early osteoclastogenesis by receptor activation of nuclear factor κB ligand (RANKL), an osteoclastogenic cytokine. Here, we report the novel enzymatic function of JMJD5, which regulates NFATc1 protein stability. Among the tested Jumonji C (JmjC) domain-containing proteins, decreased mRNA expression levels during osteoclastogenesis were found for JMJD5 in RAW264 cells stimulated by RANKL. To examine the functional role of JMJD5 in osteoclast differentiation, we established stable JMJD5 knockdown cells, and osteoclast formation was assessed. Down-regulated expression of JMJD5 led to accelerated osteoclast formation together with induction of several osteoclast-specific genes such as Ctsk and DC-STAMP, suggesting that JMJD5 is a negative regulator in osteoclast differentiation. Although JMJD5 was recently reported as a histone demethylase for histone H3K36me2, no histone demethylase activity was detected in JMJD5 in vitro or in living cells, even for other methylated histone residues. Instead, JMJD5 co-repressed transcriptional activity by destabilizing NFATc1 protein. Protein hydroxylase activity mediated by the JmjC domain in JMJD5 was required for the observed functions of JMJD5. JMJD5 induced the association of hydroxylated NFATc1 with the E3 ubiquitin ligase Von Hippel-Lindau tumor suppressor (VHL), thereby presumably facilitating proteasomal degradation of NFATc1 via ubiquitination. Taken together, the present study demonstrated that JMJD5 is a post-translational co-repressor for NFATc1 that attenuates osteoclastogenesis.

Jmjd5, an H3K36me2 histone demethylase, modulates embryonic cell proliferation through the regulation of Cdkn1a expression.

Covalent modifications of histones play an important role in chromatin architecture and dynamics. In particular, histone lysine methylation is important for transcriptional control during diverse biological processes. The nuclear protein Jmjd5 (also called Kdm8) is a histone lysine demethylase that contains a JmjC domain in the C-terminal region. In this study, we have generated Jmjd5-deficient mice (Jmjd5(Δ)(/)(Δ)) to investigate the in vivo function of Jmjd5. Jmjd5(Δ)(/)(Δ) embryos showed severe growth retardation, resulting in embryonic lethality at the mid-gestation stage. Mouse embryonic fibroblasts (MEFs) derived from Jmjd5 hypomorphic embryos (Jmjd5(neo/neo)) also showed the growth defect. Quantitative PCR analysis of various cell cycle regulators indicated that only Cdkn1a expression was upregulated in Jmjd5(neo/neo) MEFs and Jmjd5(Δ)(/)(Δ) embryos. A knockdown assay with Cdkn1a-specific small interfering RNAs revealed that the growth defect of Jmjd5(neo/neo) MEFs was significantly rescued. In addition, a genetic study using Jmjd5(Δ)(/)(Δ); Cdkn1a(Δ)(/)(Δ) double-knockout mice showed that the growth retardation of Jmjd5(Δ)(/)(Δ) embryos was partially rescued by Cdkn1a deficiency. Chromatin immunoprecipitation analysis showed that increased di-methylated lysine 36 of histone H3 (H3K36me2) and reduced recruitment of endogenous Jmjd5 were detected in the transcribed regions of Cdkn1a in Jmjd5(neo/neo) MEFs. Taken together, these results suggest that Jmjd5 physiologically moderates embryonic cell proliferation through the epigenetic control of Cdkn1a expression.

In vitro generation of HSC-like cells from murine ESCs/iPSCs by enforced expression of LIM-homeobox transcription factor Lhx2.

Identification of genes involved in in vitro differentiation induction of embryonic stem cells (ESCs) into hematopoietic stem cells (HSCs) has been challenged during last decade. To date, a homeobox transcription factor Hoxb4 has been only demonstrated to possess such an effect in mice. Here, we show that HSC-like cells were efficiently induced from mouse ESCs by enforced expression of Lhx2, a LIM-homeobox transcription factor. Transduction of Lhx2 into ESC-derived mesodermal cells resulted in robust differentiation of c-Kit(+)/Sca-1(+)/Lineage(-) (KSL) cells in vitro. The KSL cell induction frequency was superior to the case of Hoxb4. Furthermore, transplantation of Lhx2-transduced hematopoietic cells into lethally irradiated mice resulted in multilineage repopulation of hematopoietic cells over 4 months. Transduction of Lhx2 into induced pluripotent stem cells (iPSCs) was also effective in generating KSL cells in vitro, as well as HSC-like activities in vivo. These results demonstrate that ectopic expression of Lhx2 confers an in vivo engrafting capacity to ESC/iPSC-derived hematopoietic cells and in vivo behavior of iPSC-derived hematopoietic cells is almost identical to that of ESC-derived cells.

Oncostatin M deficiency leads to thymic hypoplasia, accumulation of apoptotic thymocytes and glomerulonephritis.

Oncostatin M (OSM) has been implicated in immune regulation, though its precise role remains elusive. Here we show that OSM plays a crucial role in the prevention of autoimmune diseases. OSM-deficient mice showed normal development of T cells, B cells and DC; however, their thymus showed hypoplasia and altered medullary structure. Autoantibodies against dsDNA accumulated and glomerulonephritis developed in aged OSM-deficient mice. Apoptotic cells accumulated in the thymus of OSM-deficient mice, and the administration of dexamethasone in young OSM-deficient mice resulted in the massive accumulation of apoptotic thymocytes and production of autoantibodies. These results suggest that OSM plays a key role in the prevention of autoimmune disease by regulating the clearance of apoptotic thymocytes.

meis1 regulates the development of endothelial cells in zebrafish.

The differentiation of endothelial cells is tightly connected with the formation of blood vessels during vertebrate development. The signaling pathways mediated by vascular endothelial growth factor (vegf) are required for these processes. Here we show that a proto-oncogene, meis1, plays important roles in the vascular development in zebrafish. Knockdown of meis1 by anti-sense meis1 morpholino (meis1 MO) led to the impairment of intersegmental vessel (ISV) formation. In meis1 morphants, the expression of an artery marker was reduced in dorsal aorta (DA), and the expression of vein markers was expanded in DA and posterior cardinal vein (PCV), suggesting the defects on artery development. Furthermore, the expression of vegf receptor, flk1, was significantly decreased in these embryos. Interestingly, flk1 MO-injected embryos exhibited similar defects as meis1 morphants. Thus, these results implicate that meis1 is a novel regulator involved in endothelial cell development, presumably affecting the vegf signaling pathway.

Oncostatin m maintains the hematopoietic microenvironment and retains hematopoietic progenitors in the bone marrow.

Bone marrow (BM) functions as the primary hematopoietic tissue throughout adult life by providing a microenvironment for the proliferation, differentiation, and retention of hematopoietic stem cells and progenitors. We describe novel roles for oncostatin M (OSM) in the BM hematopoietic microenvironment. Hematopoietic progenitor activity in OSM-deficient mice was reduced in BM but elevated in the spleen and peripheral blood. The level of circulating granulocyte colony-stimulating factor (G-CSF) was increased, whereas that of stromal cell-derived factor 1 (SDF-1) was decreased in OSM-deficient mice. Moreover, the ability of OSM-deficient BM stromal cells to support hematopoiesis in vitro was significantly reduced. These results indicate that OSM plays a unique role in hematopoiesis by maintaining the proper microenvironment for BM hematopoiesis; it also retains hematopoietic progenitors in BM by regulating G-CSF and SDF-1 levels.

Tumor suppressor gene identification using retroviral insertional mutagenesis in Blm-deficient mice.

Retroviral insertional mutagenesis preferentially identifies oncogenes rather than tumor suppressor (TS) genes, presumably because a single retroviral-induced mutation is sufficient to activate an oncogene and initiate a tumor, whereas two mutations are needed to inactivate a TS gene. Here we show that TS genes can be identified by insertional mutagenesis when the screens are performed in Blm-deficient backgrounds. Blm-deficient mice, like Bloom syndrome patients, have increased frequencies of mitotic recombination owing to a mutation in the RecQ protein-like-3 helicase gene. This increased mitotic recombination increases the likelihood that an insertional mutation in one allele of a TS gene will become homozygoused by non-sister chromatid exchange and the homozygosity of the insertion provides a marker for identifying the TS gene. We also show that known as well as novel TS genes can be identified by insertional mutagenesis in Blm-deficient mice and identify two JmjC family proteins that contribute to genome stability in species as evolutionarily diverse as mammals and Caenorhabditis elegans.

Essential function of oncostatin m in nociceptive neurons of dorsal root ganglia.

Oncostatin M (OSM) is a member of the interleukin-6 family of cytokines, and we have reported previously that the murine OSM receptor beta subunit (OSMR) was expressed in some neurons in the adult trigeminal and dorsal root ganglia (DRGs) and in the perineonatal hypoglossal nucleus. In the present study, we investigated the development of OSMR-positive neurons of DRGs in OSM-deficient mice. In situ hybridization revealed that OSMR-positive neurons in DRGs began to appear at postnatal day 0 (P0) and reached the adult level at P14. In the DRGs of the OSM-deficient mice, vanilloid receptor 1 (VR1)- and P2X3-positive small-sized neurons were significantly decreased. In addition, OSMR-positive neurons decreased, resulting in the reduced number of VR1/P2X3/OSMR-triple positive neurons. OSM-deficient mice displayed significantly reduced noxious responses in models of acute thermal, mechanical, chemical, and visceral pain. Thus, OSM plays an essential role in the development of a subtype of nociceptive neurons in the DRGs.

Macrophage colony stimulating factor modulates the development of hematopoiesis by stimulating the differentiation of endothelial cells in the AGM region.

Definitive hematopoietic stem cells arise in the aorta-gonad-mesonephros (AGM) region from hemangioblasts, common precursors for hematopoietic and endothelial cells. Previously, we showed that multipotential hematopoietic progenitors and endothelial cells were massively produced in primary culture of the AGM region in the presence of oncostatin M. Here we describe a role for macrophage-colony-stimulating factor (M-CSF) in the development of hematopoietic and endothelial cells in AGM culture. The number of hematopoietic progenitors including multipotential cells was significantly increased in the AGM culture of op/op embryos. The addition of M-CSF to op/op AGM culture decreased colony-forming unit (CFU)-GEMM, granulocyte macrophage-CFU, and erythroid-CFU, but it increased CFU-M. On the other hand, the number of cells expressing endothelial markers, vascular endothelial-cadherin, intercellular adhesion molecule 2, and Flk-1 was reduced in op/op AGM culture. The M-CSF receptor was expressed in PCLP1(+)CD45(-) cells, the precursors of endothelial cells, and M-CSF up-regulated the expression of more mature endothelial cell markers-VCAM-1, PECAM-1, and E-selectin-in PCLP1(+)CD45(-) cells. These results suggest that M-CSF modulates the development of hematopoiesis by stimulating the differentiation of PCLP-1(+)CD45(-) cells to endothelial cells in the AGM region.