Yingmin Liang

Notch Signaling Determines the M1 versus M2 Polarization of Macrophages in Antitumor Immune Responses

Macrophages are important tumor-infiltrating cells and play pivotal roles in tumor growth and metastasis. Macrophages participate in immune responses to tumors in a polarized manner: classic M1 macrophages produce interleukin (IL) 12 to promote tumoricidal responses, whereas M2 macrophages produce IL10 and help tumor progression. The mechanisms governing macrophage polarization are unclear. Here, we show that the M2-like tumor-associated macrophages (TAM) have a lower level of Notch pathway activation in mouse tumor models. Forced activation of Notch signaling increased M1 macrophages which produce IL12, no matter whether M1 or M2 inducers were applied. When Notch signaling was blocked, the M1 inducers induced M2 response in the expense of M1. Macrophages deficient in canonical Notch signaling showed TAM phenotypes. Forced activation of Notch signaling in macrophages enhanced their antitumor capacity. We further show that RBP-J-mediated Notch signaling regulates the M1 versus M2 polarization through SOCS3. Therefore, Notch signaling plays critical roles in the determination of M1 versus M2 polarization of macrophages, and compromised Notch pathway activation will lead to the M2-like TAMs. These results provide new insights into the molecular mechanisms of macrophage polarization and shed light on new therapies for cancers through the modulation of macrophage polarization through the Notch signaling.

RBP-J, the transcription factor downstream of Notch receptors, is essential for the maintenance of vascular homeostasis in adult mice

In adults, angiogenic abnormalities are involved in not only tumor growth but several human inherited diseases as well. It is unclear, however, concerning how the normal vascular structure is maintained and how angiogenesis is initiated in normal adults. Using the Cre‐LoxP‐mediated conditional gene deletion, we show in the present study that in adult mice disruption of the transcription factor recombination signal‐binding protein Jκ (RBP‐J) in endothelial cells strikingly induced spontaneous angiogenesis in multiple tissues, including retina and cornea, as well as in internal organs, such as liver and lung. In a choroidal neovascularization model, which mimics the angiogenic process in tumor growth and age‐related macular degeneration, RBP‐J deficiency induced a more intensive angiogenic response to injury. This could be transmitted by bone marrow, indicating that RBP‐J could modulate bone marrow‐derived endothelial progenitor cells in adult angiogenesis. In addition, in the absence of RBP‐J, proliferation of endothelial cells increased significantly, leading to accumulative vessel outgrowth. These findings suggest that in adults RBP‐J‐mediated Notch signaling may play an essential role in the maintenance of vascular homeostasis by repressing endothelial cell proliferation.—Dou, G.‐R., Wang, Y.‐C., Hu, X.‐B., Hou, L.‐H., Wang, C.‐M., Xu, J.‐F., Wang, Y.‐S., Liang, Y.‐M., Yao, L.‐B., Yang, A.‐G., Han, H. RBP‐J, the transcription factor downstream of Notch receptors, is essential for the maintenance of vascular homeostasis in adult mice. FASEB J. 22, 1606–1617 (2008)

Lipopolysaccharide-induced Maturation of Bone Marrow-derived Dendritic Cells Is Regulated by Notch Signaling through the Up-regulation of CXCR4

Dendritic cells (DCs) are professional antigen presenting cells to initiate immune response against pathogens, but mechanisms controlling the maturation of DCs are unclear. Here we report that, in the absence of recombination signal binding protein-Jκ (RBP-J, the transcription factor mediating Notch signaling), lipopolysaccharide-stimulated monocyte-derived DCs are arrested at a developmental stage with few dendrites, low major histocompatibility complex II (MHC II) expression, and reduced motility and antigen presentation ability. RBP-J null DCs had lower expression of CXCR4. Transduction with a CXCR4-expressing lentivirus rescued developmental arrest of RBP-J-deficient DCs. Activation of Notch signaling in DCs up-regulated CXCR4 expression and increased the outgrowth of dendrites and the expression of MHC II. These effects were abrogated by a CXCR4 inhibitor. Therefore, Notch signaling is essential for DCs to transit from a dendritelowMHC IIlow immature state into a dendritehighMHC IIhigh mature state, during the lipopolysaccharide-induced DC maturation, most likely through the up-regulation of CXCR4.

Disruption of the transcription factor recombination signal‐binding protein‐Jκ (RBP‐J) leads to veno‐occlusive disease and interfered liver regeneration in mice

Liver sinusoid (LS) endothelial cells (LSECs) support hepatocytes in resting livers and proliferate during liver regeneration to revascularize regenerated liver parenchyma. We report that recombination signal‐binding protein‐Jκ (RBP‐J), the critical transcription factor mediating Notch signaling, regulates both resting and regenerating LSECs. Conditional deletion of RBP‐J resulted in LSEC proliferation and a veno‐occlusive disease–like phenotype in the liver, as manifested by liver congestion, deposition of fibrin‐like materials in LSs, edema in the space of Disse, and increased apoptosis of hepatocytes. Regeneration of liver was remarkably impaired, with reduced LSEC proliferation and destroyed sinusoidal structure. LSEC degeneration was obvious in the regenerating liver of RBP‐J–deficient mice, with some LSECs losing cytoplasm, and organelles protruding into the remnant plasma‐membrane of LSs to hamper the microcirculation and intensify veno‐occlusive disease during liver regeneration. Hepatocytes were also degenerative, as shown by dilated endoplasmic reticulum, decreased proliferation, and increased apoptosis during liver regeneration. Molecular analyses revealed that the dynamic expression of several related molecules—such as vascular endothelial growth factor, vascular endothelial growth factor receptors 1 and 2, interleukin‐6, and hepatocyte growth factor—was disturbed. Conclusion: Notch/RBP‐J signaling may play dual roles in LSECs: in resting liver it represses proliferation, and in regenerating liver it supports proliferation and functional differentiation. (HEPATOLOGY 2009;49:268‐277.)

Myc induced miR-144/451 contributes to the acquired imatinib resistance in chronic myelogenous leukemia cell K562

Imatinib resistance remains the big hurdle for CML therapy. Previous study reveals that c-myc is important for bcr-abl CML cell proliferation, while its role in imatinib resistance is largely unknown. In this study, we first found that c-myc expression is upregulated in imatinib resistant K562R cells, which in turn enhances the expression of miR-144/451. Knockdown of c-myc or restoration of miR-144/451 in the K562R cells sensitizes K562R cells to imatinib therapy. Our study here reveals an regulatory pathway between myc and miR-144/451 and highlights that targeting either myc or miR-144/451 might be valuable for eliminating the imatinib resistant CML cells.

Notch-RBP-J Signaling Regulates the Mobilization and Function of Endothelial Progenitor Cells by Dynamic Modulation of CXCR4 Expression in Mice

Bone marrow (BM)-derived endothelial progenitor cells (EPC) have therapeutic potentials in promoting tissue regeneration, but how these cells are modulated in vivo has been elusive. Here, we report that RBP-J, the critical transcription factor mediating Notch signaling, modulates EPC through CXCR4. In a mouse partial hepatectomy (PHx) model, RBP-J deficient EPC showed attenuated capacities of homing and facilitating liver regeneration. In resting mice, the conditional deletion of RBP-J led to a decrease of BM EPC, with a concomitant increase of EPC in the peripheral blood. This was accompanied by a down-regulation of CXCR4 on EPC in BM, although CXCR4 expression on EPC in the circulation was up-regulated in the absence of RBP-J. PHx in RBP-J deficient mice induced stronger EPC mobilization. In vitro, RBP-J deficient EPC showed lowered capacities of adhering, migrating, and forming vessel-like structures in three-dimensional cultures. Over-expression of CXCR4 could at least rescue the defects in vessel formation by the RBP-J deficient EPC. These data suggested that the RBP-J-mediated Notch signaling regulated EPC mobilization and function, at least partially through dynamic modulation of CXCR4 expression. Our findings not only provide new insights into the regulation of EPC, but also have implications for clinical therapies using EPC in diseases.

The PcG protein HPC2 inhibits RBP-J-mediated transcription by interacting with LIM protein KyoT2

The DNA‐binding protein recombination signal‐binding protein‐Jk (RBP‐J) plays a key role in transcriptional regulation by targeting the intracellular domain of Notch (NIC) and the Epstein–Barr virus nuclear antigen 2 (EBNA2) to specific promoters. In the absence of the Notch signaling, RBP‐J acts as a transcriptional suppressor through recruiting co‐suppressors such as histone deacetylase (HDAC). KyoT2 is a LIM domain protein that suppresses the RBP‐J‐mediated transcriptional activation. In the current study, we show that the polycomb group (PcG) protein HPC2, which functions as a transcriptional suppressor, is a candidate of KyoT2‐binding proteins. To confirm the physical and functional interaction between KyoT2 and HPC2, we carried out yeast two‐hybrid, GST‐pull down, co‐immunoprecipitation, as well as mammalian two‐hybrid assays. Our results showed HPC2 and KyoT2 interacted both in vitro and in vivo, probably through the C‐terminal fragment of HPC2 and LIM domains of KyoT2. In addition, we also found that overexpression of HPC2, not only inhibited transactivation of a RBP‐J‐dependent promoter by NIC, but also transactivation by RBP‐J–VP16, a constitutively active form of RBP‐J. Taken together, our results suggested that KyoT2 might inhibit the RBP‐J‐mediated transactivation through NIC by recruiting co‐suppressors such as HPC2.

Notch signaling inhibits the growth of the human chronic myeloid leukemia cell line K562

Notch signaling functions in the development of some types of leukemia and lymphoma, but the relationship between Notch signaling and chronic myeloid leukemia (CML) remains to be elucidated. In this study, we examined the expression of Notch receptors and ligands in the human CML cell line K562. When the active form of Notch1, the Notch intra-cellular domain (NIC), was over-expressed in K562, the proliferation of K562 was mildly but significantly inhibited, accompanied by increased Hes1 mRNA level. On the other hand, when Notch signaling was attenuated by over-expression of a dominant-negative RBP-J, RBP-J(R218H), in K562 cells, the proliferation of K562 was increased. Moreover, we found that activation of Notch signaling inhibited while repression of Notch signaling promoted the colony-forming activity of K562 cells. We examined cell cycle-related molecules in K562 transfected with NIC or RBP-J(R218H), and found that the protein level of the retinoblastoma gene product (the Rb protein) was induced in K562 expressing NIC, and down-regulated in K562 expressing RBP-J(R218H). These data suggest that the Notch signaling may function as a tumor inhibitor in human CML cells.

KyoT3, an isoform of murine FHL1, associates with the transcription factor RBP-J and represses the RBP-J-mediated transactivation

Previously, we have shown that KyoT2, an isoform of the four and a half LIM domain protein 1 (FHL1), modulates Notch signaling via repressing RBP-J-mediated transactivation. In this study, we investigated the effect of another isoform of FHL1, KyoT3, on transactivation of a RBP-J-dependent promoter. We found that KyoT3 was expressed widely in a variety of tissues. By constructing EGFP fusion proteins, we showed that KyoT3 locates preferentially in nucleus. KyoT3 interacted with RBP-J, as shown by co-immunoprecipitation assays. Moreover, we demonstrated by a reporter assay that KyoT3 repressed transactivation of a RBP-J-dependent promoter, which was activated by both the Notch intracellular domain and Epstein-Barr virus nuclear antigen 2, an EB virus-encoded oncoprotein. These results suggest a multi-elemental control of the Notch signaling pathway, which is critical for cell differentiation in development.

Mint Represses Transactivation of the Type II Collagen Gene Enhancer through Interaction with αA-crystallin-binding Protein 1

Collagen type II is an extracellular matrix protein important for cartilage and bone formation, and its expression is controlled by multiple cis- and trans-acting elements, including the zinc finger transcription factor αA-crystallin-binding protein 1 (CRYBP1). Here we show that MSX2-interacting nuclear target protein (MINT), a conserved transcriptional repressor, associates with CRYBP1 and negatively regulates the transactivation of the collagen type II gene (Col2a1) enhancer. We identified CRYBP1 as a binding partner of MINT by screening a mouse embryonic cDNA library using the yeast two-hybrid system. We demonstrated that the C terminus of MINT interacts with the C terminus of CRYBP1 using the mammalian cell two-hybrid assay, glutathione S-transferase pull-down, and co-immunoprecipitation analyses. Furthermore, MINT and CRYBP1 form a complex on the Col2a1 enhancer, as shown by chromatin immunoprecipitation and gel shift assays. In the presence of CRYBP1, overexpression of MINT or its C-terminal fragment in cells repressed a reporter construct driven by the Col2a1 enhancer elements. This transcription repression is dependent on histone deacetylase, the main co-repressor recruited by MINT. The present study shows that MINT is involved in CRYBP1-mediated Col2a1 gene repression and may play a role in regulation of cartilage development.