Hong-Yan Qin

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.

Canonical notch pathway protects hepatocytes from ischemia/reperfusion injury in mice by repressing reactive oxygen species production through JAK2/STAT3 signaling

Hepatic ischemia/reperfusion (I/R) injury is initiated by reactive oxygen species (ROS) accumulated during the early reperfusion phase after ischemia, but cellular mechanisms controlling ROS production and scavenging have not been fully understood. In this study, we show that blocking Notch signal by knockout of the transcription factor RBP‐J or a pharmacological inhibitor led to aggravated hepatic I/R injury, as manifested by deteriorated liver function and increased apoptosis, necrosis, and inflammation, both in vitro and in vivo. Interruption of Notch signaling resulted in increased intracellular ROS in hepatocytes, and a ROS scavenger cured exacerbated hepatic I/R injury after Notch signaling blockade, suggesting that Notch signal deficiency aggravated I/R injury through increased ROS levels. Notch signal blockade resulted in down‐regulation of Hes5, leading to reduced formation of the Hes5‐STAT3 complex and hypophosphorylation of STAT3, which further attenuated manganese superoxide dismutase (MnSOD) expression and increased ROS and apoptosis. Indeed, overexpression of a constitutively active STAT3 rescued MnSOD expression and I/R injury–induced apoptosis in the absence of Notch signaling. Finally, forced Notch activation by ligand stimulation or Hes5 overexpression reduced intracellular ROS and protected hepatocytes from apoptosis after I/R injury through the activation of STAT3 and MnSOD expression.

Overexpressed active Notch1 induces cell growth arrest of HeLa cervical carcinoma cells

Human cervical carcinoma is one of the most common malignant tumors, but the mechanisms that orchestrate the multiple oncogenic insults required for initiation and progression are not clear. Notch signaling plays a critical role in maintaining the balance between cell proliferation, differentiation, and apoptosis, but perturbed Notch signaling may contribute to tumorigenesis. We now show that Notch1 is detected in all cervical cancer, including advanced diseases. We also constitutively overexpressed active Notch1 in human cervical carcinoma to explore the effects of Notch1 signaling on human cervical carcinoma cell growth and to investigate the underlying molecular mechanisms. The signaling may participate in the development of human cervical carcinoma cells, but overexpressed active Notch1 inhibits their growth through induction of cell cycle arrest. Increased Notch1 signaling induced a downmodulation of human papillomavirus transcription through suppression of activator protein (AP)-1 activity by upregulation of c-Jun and the decreased expression of c-Fos. Thus, Notch1 signaling plays a key role and exerts dual effects, functioning in context-specific manner

Forced Activation of Notch in Macrophages Represses Tumor Growth by Upregulating miR-125a and Disabling Tumor-Associated Macrophages

Tumor-associated macrophages (TAM) contribute greatly to hallmarks of cancer. Notch blockade was shown to arrest TAM differentiation, but the precise role and underlying mechanisms require elucidation. In this study, we employed a transgenic mouse model in which the Notch1 intracellular domain (NIC) is activated conditionally to define the effects of active Notch1 signaling in macrophages. NIC overexpression had no effect on TAM differentiation, but it abrogated TAM function, leading to repressed growth of transplanted tumors. Macrophage miRNA profiling identified a novel downstream mediator of Notch signaling, miR-125a, which was upregulated through an RBP-J-binding site at the first intronic enhancer of the host gene Spaca6A. miR-125a functioned downstream of Notch signaling to reciprocally influence polarization of M1 and M2 macrophages by regulating factor inhibiting hypoxia inducible factor-1α and IRF4, respectively. Notably, macrophages transfected with miR-125a mimetics increased phagocytic activity and repressed tumor growth by remodeling the immune microenvironment. We also identified a positive feedback loop for miR-125a expression mediated by RYBP and YY1. Taken together, our results showed that Notch signaling not only supported the differentiation of TAM but also antagonized their protumorigenic function through miR-125a. Targeting this miRNA may reprogram macrophages in the tumor microenvironment and restore their antitumor potential.

Transcription factor RBP-J-mediated signaling represses the differentiation of neural stem cells into intermediate neural progenitors.

Notch signaling is critical for multiple aspects of neurogenesis, but how it regulates the proliferation and differentiation of neural stem cells (NSCs) and intermediate neural progenitors (INPs) has not been well elucidated, especially in vivo. In this study, we conditionally ablated the transcription factor RBP-J, which mediates signaling from all four mammalian Notch receptors, in the basal forebrain and ventral midbrain using the RBP-J-floxed mouse and a newly established Nestin-Cre mouse. We found that at early stage of neurogenesis (E11.5), the frequency of neurospheres increased significantly in the RBP-J-inactivated regions. The majority of the RBP-J deficient neurospheres were composed of INPs, suggesting the precocious differentiation of NSCs into INPs. Meanwhile, neuronal differentiation was reduced in the same regions at E11.5, inconsistent with the precocious differentiation phenotype in most Notch-related mutants. At late neurogenic stages (E17.5 and neonatal), as expected from precociously exhausted NSC pool, neurosphere frequency and NSCs decreased in the RBP-J-ablated regions, accompanied by a significant increase of both neurons and glial cells. These results indicated that the RBP-J-mediated signaling might inhibit the differentiation of NSCs into INPs and support the generation of certain early born neurons at early neurogenic stages.

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.

Myeloid‐specific disruption of recombination signal binding protein Jκ ameliorates hepatic fibrosis by attenuating inflammation through cylindromatosis in mice

Macrophages play multidimensional roles in hepatic fibrosis, but their control has not been fully understood. The Notch pathway mediated by recombination signal binding protein Jκ (RBP‐J), the transcription factor transactivated by signals from four mammalian Notch receptors, is implicated in macrophage activation and plasticity. In this study, by using mouse hepatic fibrosis models, we show that myeloid‐specific disruption of RBP‐J resulted in attenuated fibrosis. The activation of hepatic stellate cells and production of profibrotic factors including platelet‐derived growth factor (PDGF)‐B and transforming growth factor beta1 (TGF‐β1) reduced significantly in myeloid‐specific RBP‐J deficient mice. The infiltration of inflammatory cells and production of proinflammatory factors were reduced in liver of myeloid‐specific RBP‐J‐deficient mice during fibrosis. In RBP‐J‐deficient macrophages, the nuclear factor kappa B (NF‐κB) activation was remarkably attenuated as compared with the control. This could be attributed to the up‐regulation of cylindromatosis (CYLD), a negative regulator of NF‐κB, in Notch signal‐compromised macrophages, because the knockdown of CYLD in RBP‐J‐deficient macrophages or overexpression of p65 in RBP‐J knockdown cells both restored NF‐κB activation and the production of proinflammatory and/or profibrotic factors by macrophages. In human hepatic fibrosis biopsies, stronger Notch activation is correlated with more severe fibrosis, which is accompanied by a lower level of CYLD but irrespective of etiological reasons. Conclusion: RBP‐J‐mediated Notch signaling is required for macrophages to promote hepatic fibrosis by up‐regulation of NF‐κB activation through CYLD. (Hepatology 2015;61:303–314)

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.

Overexpression of Notch ligand Dll1 in B16 melanoma cells leads to reduced tumor growth due to attenuated vascularization.

Notch signaling plays an important role in vascular development and tumor angiogenesis. It has been shown that disruption of Dll4-triggered Notch signal activation effectively inhibits tumor growth, but this treatment also results in the formation of vascular neoplasms. In this study, we investigate the effects of over-expressing Notch ligand Dll1 in B16 melanoma cells on tumor cell proliferation and tumor growth in vitro and in vivo. Our results showed that over-expression of Dll1 could activate Notch signaling in tumor cells, and promote tumor cell proliferation in vitro. In contrast, growth of Dll1-over-expressing tumors in vivo was reduced, due to abnormal tumor vessel formation. Impaired tumor vasculature enhanced hypoxia and necrosis in tumor tissues, leading to retarded tumor growth. These results suggest that activation of Notch signaling may serve as an anti-angiogenesis strategy in the treatment of malignant tumors.

Monocyte to macrophage differentiation-associated (MMD) positively regulates ERK and Akt activation and TNF-α and NO production in macrophages

Macrophage activation is modulated by both environmental cues and endogenous programs. In the present study, we investigated the role of a PAQR family protein, monocyte to macrophage differentiation-associated (MMD), in macrophage activation and unveiled its underlying molecular mechanism. Our results showed that while MMD expression could be detected in all tissues examined, its expression level is significantly up-regulated upon monocyte differentiation. Within cells, EGFP–MMD fusion protein could be co-localized to endoplasmic reticulum, mitochondria, Golgi apparatus, but not lysosomes and cytoplasm. MMD expression is up-regulated in macrophages after LPS stimulation, and this might be modulated by RBP-J, the critical transcription factor of Notch signaling. Overexpression of MMD in macrophages increased the production of TNF-α and NO upon LPS stimulation. We found that MMD overexpression enhanced ERK1/2 and Akt phosphorylation in macrophages after LPS stimulation. Blocking Erk or Akt by pharmacological agent reduced TNF-α or NO production in MMD-overexpressing macrophages, respectively. These results suggested that MMD modulates TNF-α and NO production in macrophages, and this process might involves Erk or Akt.