Gonghui Li

Neutropenia with impaired host defense against microbial infection in mice lacking androgen receptor

Neutrophils, the major phagocytes that form the first line of cell-mediated defense against microbial infection, are produced in the bone marrow and released into the circulation in response to granulocyte-colony stimulating factor (G-CSF). Here, we report that androgen receptor knockout (ARKO) mice are neutropenic and susceptible to acute bacterial infection, whereas castration only results in moderate neutrophil reduction in mice and humans. Androgen supplement can restore neutrophil counts via stabilizing AR in castrated mice, but not in ARKO and testicular feminization mutant (Tfm) mice. Our results show that deletion of the AR gene does not influence myeloid lineage commitment, but significantly reduces the proliferative activity of neutrophil precursors and retards neutrophil maturation. CXCR2-dependent migration is also decreased in ARKO neutrophils as compared with wild-type controls. G-CSF is unable to delay apoptosis in ARKO neutrophils, and ARKO mice show a poor granulopoietic response to exogenous G-CSF injection. In addition, AR can restore G-CSF–dependent granulocytic differentiation upon transduction into ARKO progenitors. We further found that AR augments G-CSF signaling by activating extracellular signal-regulated kinase 1/2 and also by sustaining Stat3 activity via diminishing the inhibitory binding of PIAS3 to Stat3. Collectively, our findings demonstrate an essential role for AR in granulopoiesis and host defense against microbial infection.

Tissue Prostate-Specific Antigen Facilitates Refractory Prostate Tumor Progression via Enhancing ARA70-Regulated Androgen Receptor Transactivation

Despite being well recognized as the best biomarker for prostate cancer, pathophysiologic roles of prostate-specific antigen (PSA) remain unclear. We report here that tissue PSA may be involved in the hormone-refractory prostate cancer progression. Histologic analyses show that the increased tissue PSA levels are correlated with lower cell apoptosis index and higher cell proliferation rate in hormone-refractory tumor specimens. By stably transfecting PSA cDNA into various prostate cancer cell lines, we found that PSA could promote the growth of androgen receptor (AR)-positive CWR22rv1 and high-passage LNCaP (hormone-refractory prostate cancer cells) but not that of AR-negative PC-3 and DU145 cells. Surprisingly, the protease activity of PSA is not crucial for PSA to stimulate growth and promote AR transactivation. We further showed that increased PSA could enhance ARA70-induced AR transactivation via modulating the p53 pathway that results in the decreased apoptosis and increased cell proliferation in prostate cancer cells. Knockdown of PSA in LNCaP and CWR22rv1 cells causes cell apoptosis and cell growth arrest at the G(1) phase. In vitro colony formation assay and in vivo xenografted tumor results showed the suppression of prostate cancer growth via targeting PSA expression. Collectively, our findings suggest that, in addition to being a biomarker, PSA may also become a new potential therapeutic target for prostate cancer. PSA small interfering RNA or smaller molecules that can degrade PSA protein may be developed as alternative approaches to treat the prostate cancer.

Increased Chemosensitivity via Targeting Testicular Nuclear Receptor 4 (TR4)-Oct4-Interleukin 1 Receptor Antagonist (IL1Ra) Axis in Prostate Cancer CD133+ Stem/Progenitor Cells to Battle Prostate Cancer

Background: PCa stem/progenitor cells develop higher chemoresistance. Results: High TR4 levels in PCa stem/progenitor cells were shown to be critical in conferring chemoresistance to these cells. Conclusion: TR4-Oct4-IL1Ra signaling is important in conferring chemoresistance to PCa stem/progenitor cells. Significance: This finding suggests that targeting TR4 and its downstream molecules may be a better therapeutic approach to battle PCa stem/progenitor cell-originated chemoresistance. Prostate cancer (PCa) stem/progenitor cells are known to have higher chemoresistance than non-stem/progenitor cells, but the underlying molecular mechanism remains unclear. We found the expression of testicular nuclear receptor 4 (TR4) is significantly higher in PCa CD133+ stem/progenitor cells compared with CD133− non-stem/progenitor cells. Knockdown of TR4 levels in the established PCa stem/progenitor cells and the CD133+ population of the C4-2 PCa cell line with lentiviral TR4 siRNA led to increased drug sensitivity to the two commonly used chemotherapeutic drugs, docetaxel and etoposide, judging from significantly reduced IC50 values and increased apoptosis in the TR4 knockdown cells. Mechanism dissection studies found that suppression of TR4 in these stem/progenitor cells led to down-regulation of Oct4 expression, which, in turn, down-regulated the IL-1 receptor antagonist (IL1Ra) expression. Neutralization experiments via adding these molecules into the TR4 knockdown PCa stem/progenitor cells reversed the chemoresistance, suggesting that the TR4-Oct4-IL1Ra axis may play a critical role in the development of chemoresistance in the PCa stem/progenitor cells. Together, these studies suggest that targeting TR4 may alter chemoresistance of PCa stem/progenitor cells, and this finding provides the possibility of targeting TR4 as a new and better approach to overcome the chemoresistance problem in PCa therapeutics.

A new prostate cancer therapeutic approach: Combination of androgen ablation with COX‐2 inhibitor

Prostate cancer is initially responsive to hormonal therapy, but cancers inevitably progress in an androgen‐independent fashion with virtually all tumors evolving into more aggressive androgen refractory disease. Immunohistological comparisons of cyclooxygenase 2 (COX‐2) expressions in 3 pairs of prostate cancer patients before and after the combined androgen blockade (CAB) therapy show elevated COX‐2 expressions. This observation from clinical specimens is further supported by in vitro laboratory data using human prostate cancer cells in which the antiandrogen hydroxyflutamide (HF) induced COX‐2 expression, and androgen suppressed COX‐2 expression. By applying knockdown and overexpression strategies to modulate AR expression in prostate cancer cells, we confirmed that androgen/AR signal suppressed, and HF induced COX‐2 expression at both protein and mRNA levels. COX‐2 promoter reporter assay indicated that the suppression of COX‐2 by androgen/AR is at the transcriptional level via modulation of NF‐κB signals. Treatment of LNCaP and LAPC4 cells with 1 μM HF in the presence of 1 nM DHT, which mimics the CAB therapy condition, promotes cell growth, and this growth induction can be suppressed via adding the COX‐2 specific inhibitor, NS398. This suggests that HF promoted prostate cancer cell growth is COX‐2 dependent and this HF‐COX‐2 activation pathway can account for one reason of CAB therapy failure. Together, these findings provide a possible explanation how CAB with antiandrogen HF therapy might fail and provide a potential new therapeutic approach to battle prostate cancer via combination of CAB therapy with COX‐2 inhibitor(s).

Premature aging with impaired oxidative stress defense in mice lacking TR4

Early studies suggest that TR4 nuclear receptor is a key transcriptional factor regulating various biological activities, including reproduction, cerebella development, and metabolism. Here we report that mice lacking TR4 (TR4(-/-)) exhibited increasing genome instability and defective oxidative stress defense, which are associated with premature aging phenotypes. At the cellular level, we observed rapid cellular growth arrest and less resistance to oxidative stress and DNA damage in TR4(-/-) mouse embryonic fibroblasts (MEFs) in vitro. Restoring TR4 or supplying the antioxidant N-acetyl-l-cysteine (NAC) to TR4(-/-) MEFs reduced the DNA damage and slowed down cellular growth arrest. Focused qPCR array revealed alteration of gene profiles in the DNA damage response (DDR) and anti-reactive oxygen species (ROS) pathways in TR4(-/-) MEFs, which further supports the hypothesis that the premature aging in TR4(-/-) mice might stem from oxidative DNA damage caused by increased oxidative stress or compromised genome integrity. Together, our finding identifies a novel role of TR4 in mediating the interplay between oxidative stress defense and aging.

Cisplatin enhances NK cells immunotherapy efficacy to suppress HCC progression via altering the androgen receptor (AR)-ULBP2 signals

The aim of this study is to investigate the influence of cisplatin on the efficacy of natural killer (NK) cells immunotherapy to suppress HCC progression, and provide valuable information on better application of cisplatin in clinical settings. By using in vitro cell cytotoxicity test and in vivo liver orthotopic xenograft mice model, we identified the role of cisplatin in modulating NK cells cytotoxicity. Luciferase report assay and chromatin immunoprecipitation assay were applied for mechanism dissection. Immunohistochemistry is performed for sample staining. We found cisplatin could enhance the efficacy of NK cells immunotherapy to better suppress HCC progression via altering the androgen receptor (AR)-UL16-binding protein 2 (ULBP2) signals both in vitro and in vivo. Mechanism dissection revealed that cisplatin could suppress AR expression via two distinct ways: increasing miR-34a-5p to suppress AR expression and altering the ubiquitination to accelerate the AR protein degradation. The suppressed AR might then function through up-regulating ULBP2, a natural-killer group 2 member D ligand, to enhance the cytotoxicity of NK cells. Together, these results indicated an unrecognized favoring effect of cisplatin in HCC treatment. By suppressing AR in HCC, cisplatin could up-regulate cytotoxicity of NK cells to better target HCC. This finding may provide a potential new approach to control HCC by combining traditional chemotherapy with immunotherapy.

Reduced osteoblast activity in the mice lacking TR4 nuclear receptor leads to osteoporosis

BackgroundEarly studies suggested that TR4 nuclear receptor might play important roles in the skeletal development, yet its detailed mechanism remains unclear.MethodsWe generated TR4 knockout mice and compared skeletal development with their wild type littermates. Primary bone marrow cells were cultured and we assayed bone differentiation by alkaline phosphatase and alizarin red staining. Primary calvaria were cultured and osteoblastic marker genes were detected by quantitative PCR. Luciferase reporter assays, chromatin immunoprecipitation (ChIP) assays, and electrophoretic mobility shift assays (EMSA) were performed to demonstrate TR4 can directly regulate bone differentiation marker osteocalcin.ResultsWe first found mice lacking TR4 might develop osteoporosis. We then found that osteoblast progenitor cells isolated from bone marrow of TR4 knockout mice displayed reduced osteoblast differentiation capacity and calcification. Osteoblast primary cultures from TR4 knockout mice calvaria also showed higher proliferation rates indicating lower osteoblast differentiation ability in mice after loss of TR4. Mechanism dissection found the expression of osteoblast markers genes, such as ALP, type I collagen alpha 1, osteocalcin, PTH, and PTHR was dramatically reduced in osteoblasts from TR4 knockout mice as compared to those from TR4 wild type mice. In vitro cell line studies with luciferase reporter assay, ChIP assay, and EMSA further demonstrated TR4 could bind directly to the promoter region of osteocalcin gene and induce its gene expression at the transcriptional level in a dose dependent manner.ConclusionsTogether, these results demonstrate TR4 may function as a novel transcriptional factor to play pathophysiological roles in maintaining normal osteoblast activity during the bone development and remodeling, and disruption of TR4 function may result in multiple skeletal abnormalities.

TR4 nuclear receptor promotes prostate cancer metastasis via upregulation of CCL2/CCR2 signaling.

Testicular nuclear receptor 4 (TR4) plays protective roles against oxidative stress and DNA damage and might contribute to aging. Our recent clinical tumor tissue staining results showed higher expression of TR4 in prostate cancer (PCa) patients with high Gleason scores compared to the tissues with the low Gleason scores. In vitro migration/invasion assays after manipulation of the TR4 expression in PCa cells showed that TR4 promoted PCa cells migration/invasion. Mechanism dissection found that the CCL2/CCR2 signal plays the key role in the mediation of TR4‐promoted PCa cells migration/invasion. Chromatin immunoprecipitation and Luciferase assays further confirmed TR4 modulation of CCL2 at the transcriptional level and addition of the CCR2 antagonist led to interruption of the TR4‐enhanced PCa cells migration/invasion. Finally, the orthotopic xenografted mice studies using the luciferase expressing CWR22Rv1 cells found that TR4 enhanced PCa metastasis and this increased metastasis was reversed when the CCR2 antagonist was injected into the mice. Together, these in vitro and in vivo results revealed a positive role of TR4 in PCa metastasis and demonstrated CCL2/CCR2 signaling as an important mediator in exerting TR4 action. This finding suggests that TR4 may represent a biomarker related to PCa metastasis and targeting the TR4‐CCL2/CCR2 axis may become a new therapeutic approach to battle PCa metastasis.

Mice Lacking TR4 Nuclear Receptor Develop Mitochondrial Myopathy with Deficiency in Complex I

The estimated incidence of mitochondrial diseases in humans is approximately 1:5000 to 1:10,000, whereas the molecular mechanisms for more than 50% of human mitochondrial disease cases still remain unclear. Here we report that mice lacking testicular nuclear receptor 4 (TR4(-/-)) suffered mitochondrial myopathy, and histological examination of TR4(-/-) soleus muscle revealed abnormal mitochondrial accumulation. In addition, increased serum lactate levels, decreased mitochondrial ATP production, and decreased electron transport chain complex I activity were found in TR4(-/-) mice. Restoration of TR4 into TR4(-/-) myoblasts rescued mitochondrial ATP generation capacity and complex I activity. Further real-time PCR quantification and promoter studies found TR4 could modulate complex I activity via transcriptionally regulating the complex I assembly factor NDUFAF1, and restoration of NDUFAF1 level in TR4(-/-) myoblasts increased mitochondrial ATP generation capacity and complex I activity. Together, these results suggest that TR4 plays vital roles in mitochondrial function, which may help us to better understand the pathogenesis of mitochondrial myopathy, and targeting TR4 via its ligands/activators may allow us to develop better therapeutic approaches.

Ferromagnetic structures in Mn2CoGa and Mn2CoAl doped by Co, Cu, V, and Ti

The structure and magnetic properties in doped Heusler alloys of Mn2CoGa and Mn2CoAl have been investigated by experiments and calculations. The main group elements of Ga or Al in the systems are substituted by the magnetic or non-magnetic transition metals, Co, Cu, V, and Ti. Three kinds of local ferromagnetic structures, Co-Mn-Co, Mn-Co-Mn, and Mn-Co-V, have been found. They embed in the native ferrimagnetic matrix and increase the magnetization with different increments. The Co-Mn-Co ferromagnetic structure shows the largest increment of 6.18 mu(B)/atom. In addition, interesting results for non-magnetic Cu increasing the magnetization and the V atom having a large ferromagnetic moment of about 1.0 mu(B) have been obtained. The exchange interaction energy can be increased by the newly added Co and depleted by supporting a ferromagnetic coupling in other substitution cases and showing the variation of the T-C. Our calculation of electronic structure verifies the strong d-d hybridization when the three ferromagnetic structures are achieved. It has also been found that the covalent bonding from the Ga and Al determines the generation of the local ferromagnetic structure and the tolerance for dopant content