Guiming Li

Macrophage-mediated GDNF Delivery Protects Against Dopaminergic Neurodegeneration: A Therapeutic Strategy for Parkinson's Disease

Glial cell line-derived neurotrophic factor (GDNF) has emerged as the most potent neuroprotective agent tested in experimental models for the treatment of Parkinsons disease (PD). However, its use is hindered by difficulties in delivery to the brain due to the presence of the blood-brain barrier (BBB). In order to circumvent this problem, we took advantage of the fact that bone marrow stem cell-derived macrophages are able to pass the BBB and home to sites of neuronal degeneration. Here, we report the development of a method for brain delivery of GDNF by genetically modified macrophages. Bone marrow stem cells were transduced ex vivo with lentivirus expressing a GDNF gene driven by a synthetic macrophage-specific promoter and then transplanted into recipient mice. Eight weeks after transplantation, the mice were injected with the neurotoxin, MPTP, for 7 days to induce dopaminergic neurodegeneration. Macrophage-mediated GDNF treatment dramatically ameliorated MPTP-induced degeneration of tyrosine hydroxylase (TH)-positive neurons of the substantia nigra and TH+ terminals in the striatum, stimulated axon regeneration, and reversed hypoactivity in the open field test. These results indicate that macrophage-mediated GDNF delivery is a promising strategy for developing a neuroprotective therapy for PD.Glial cell line-derived neurotrophic factor (GDNF) has emerged as the most potent neuroprotective agent tested in experimental models for the treatment of Parkinsons disease (PD). However, its use is hindered by difficulties in delivery to the brain due to the presence of the blood-brain barrier (BBB). In order to circumvent this problem, we took advantage of the fact that bone marrow stem cell-derived macrophages are able to pass the BBB and home to sites of neuronal degeneration. Here, we report the development of a method for brain delivery of GDNF by genetically modified macrophages. Bone marrow stem cells were transduced ex vivo with lentivirus expressing a GDNF gene driven by a synthetic macrophage-specific promoter and then transplanted into recipient mice. Eight weeks after transplantation, the mice were injected with the neurotoxin, MPTP, for 7 days to induce dopaminergic neurodegeneration. Macrophage-mediated GDNF treatment dramatically ameliorated MPTP-induced degeneration of tyrosine hydroxylase (TH)-positive neurons of the substantia nigra and TH(+) terminals in the striatum, stimulated axon regeneration, and reversed hypoactivity in the open field test. These results indicate that macrophage-mediated GDNF delivery is a promising strategy for developing a neuroprotective therapy for PD.

Dietary Resveratrol prevents development of high-grade prostatic intraepithelial neoplastic lesions: Involvement of SIRT1/S6K axis

SIRT1 (mammalian ortholog of the yeast silent information regulator 2) is a NAD-dependent histone deacetylase belonging to the multigene family of sirtuins. Anecdotal and epidemiologic observations provide evidence for beneficial effects of the calorie restriction mimetic resveratrol (RES), a SIRT1 activator in preventing cardiovascular diseases and cancer. Although SIRT1 possesses both tumorigenic and antitumorigenic potential, the molecular mechanisms underlying SIRT1-mediated tumor progression or inhibition are poorly understood. In this study, we investigated the role of SIRT1 in multiple human prostate cancer cell lines and prostate-specific PTEN knockout mouse model using resveratrol. Androgen-independent prostate cancer cell lines (C42B, PC3, and DU145) express higher levels of SIRT1 than androgen-responsive (LNCaP) and nontumorigenic prostate cells (RWPE-1). Resveratrol enhanced this expression without any significant effect on SIRT1 enzymatic activity. Inhibition of SIRT1 expression using shRNA enhanced cell proliferation and inhibited autophagy by repressing phosphorylation of S6K and 4E-BP1. These biologic correlates were reversed in the presence of resveratrol. Analysis of prostates from dietary intervention with resveratrol showed a significant reduction in prostate weight and reduction in the incidence of high-grade prostatic intraepithelial neoplastic (HGPIN) lesions by approximately 54% with no significant change in body weight. Consistent with the in vitro findings, resveratrol intervention in the PTEN knockout mouse model was associated with reduction in the prostatic levels of mTOR complex 1 (mTORC1) activity and increased expression of SIRT1. These data suggest that SIRT1/S6K-mediated inhibition of autophagy drives prostate tumorigenesis. Therefore, modulation of SIRT1/S6K signaling represents an effective strategy for prostate cancer prevention. Cancer Prev Res; 6(1); 27–39. ©2012 AACR.

NF-κB-Dependent Induction of Cathelicidin-Related Antimicrobial Peptide in Murine Mast Cells by Lipopolysaccharide

Background: An important aspect of the innate immune response to pathogens is the production of anti-microbial peptides such as cathelicidin-related antimicrobial peptide (CRAMP), the murine homologue of human cathelicidin LL-37. In this study, mechanisms regulating LPS-induction of CRAMP gene expression in mast cells were investigated. NF-κB and MAPK pathways were the focus of investigation. Methods: Mouse bone marrow-derived mast cells were grown in culture and stimulated with LPS. MAPKs and NF-κB were monitored by immunoblot analysis. ERK, JNK and p38 MAPK were inhibited using siRNAs or a pharmacological inhibitor. Accumulation of the p65 component of NF-κB was inhibited by siRNA and NF-κB activation was inhibited by overexpression of IκBα. MEKK2 or MEKK3 were overexpressed by transfection. The effects of all of these treatments on CRAMP gene expression were monitored by RT-PCR. Results: Inhibition of ERK, JNK or p38 MAPK had little discernible effect on LPS-inducible CRAMP gene expression. Overexpression of MEKK2 or MEKK3 likewise had little impact. However, inhibition of the accumulation of p65 NF-κB prevented LPS-induced CRAMP mRNA. An important role for NF-κB in CRAMP gene expression was confirmed by overexpression of IκBα, which reduced both basal and induced levels of CRAMP mRNA. Conclusions: NF-κB, but not MAPKs, plays an important role in LPS-mediated induction of CRAMP gene in mast cells. Defects which inhibit NF-κB activity may increase susceptibility to bacterial and viral pathogens which are sensitive to cathelicidins.

Identification of Multiple Cell Cycle Regulatory Functions of p57Kip2 in Human T Lymphocytes

The specific functions of p57Kip2 in lymphocytes have not yet been fully elucidated. In this study, it is shown that p57Kip2, which is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors, is present in the nuclei of normal resting (G0) T cells from peripheral blood and in the nuclei of the T cell-derived Jurkat cell line. Activation through the TCR results in rapid transport of cytoplasmic cyclin-dependent kinase 6 (cdk6) to nuclei, where it associates with cyclin D and p57Kip2 in active enzyme complexes. Using purified recombinant proteins, it was shown in vitro that addition of p57Kip2 protein to a mixture of cyclin D2 and cdk6 enhanced the association of the latter two proteins and resulted in phosphorylation of p57Kip2. To probe further the function of p57Kip2, Jurkat cells stably transfected with a plasmid encoding p57Kip2 under control of an inducible (tetracycline) promoter were made. Induction of p57Kip2 resulted in increased association of cdk6 with cyclin D3, without receptor-mediated T cell stimulation. The overall amounts of cdk6 and cyclin D3, and also of cdk4 and cyclin E, remained unchanged. Most notably, increased p57Kip2 levels resulted in marked inhibition of both cyclin E- and cyclin A-associated cdk2 kinase activities and a decrease in cyclin A amounts. Therefore, although facilitating activation of cdk6, the ultimate outcome of p57Kip2 induction was a decrease in DNA synthesis and cell proliferation. The results indicate that p57Kip2 is involved in the regulation of several aspects of the T cell cycle.

MEF2C regulates c‐Jun but not TNF‐α gene expression in stimulated mast cells

Mitogen‐activated protein kinase (MAPK) cascades play essential roles in the transduction of extracellular signals to cytoplasmic and nuclear effectors. The MAPK kinase kinase MEKK2 is essential for activation of c‐Jun N‐terminal kinase (JNK) and extracellular signal‐regulated kinase 5 (ERK5). These pathways are important for expression of specific cytokine genes in mast cells following cross‐linking of the high‐affinity IgE receptor (FcϵRI). A consequence of ERK5 activation is activation of the transcriptional factor myocyte enhancing factor‐2C (MEF2C), leading to increased c‐Jun expression. We have investigated the role of MEF2C activation in mast cells and demonstrated that it requires sequential activation of the signaling cascade of MEKK2‐MEK5‐ERK5. Following phosphorylation of MEF2C, activated MEF2C regulates transcription of c‐Jun but not TNF‐α. Inhibition of ERK5, MEK5 activation or activation of MEKK2‐deficient mast cells was associated with inhibition of MEF2C phosphorylation and a decrease in c‐Jun expression. Thus, these data define an activation module, MEKK2‐MEK5‐ERK5‐MEF2C in the transcriptional activation of c‐Jun in mast cells following FcϵRI cross‐linking. These results demonstrate the novel and important, MEKK2‐dependent role of MEF2C in induction of c‐Jun expression in mast cells activated through FcϵRI, a pathway distinct from that involving MEKK2‐MEK5‐ERK5 in the regulation of mast cell cytokine production.

Macrophage LXRα gene therapy ameliorates atherosclerosis as well as hypertriglyceridemia in LDLR/mice

Liver X receptors (LXRs) are implicated in the regulation of cholesterol homeostasis, inflammatory response and atherogenesis. Administration of LXR agonists inhibits the progress of atherosclerosis, and also increases plasma triglyceride levels, representing an obstacle to their use in treating this disease. The objective of this study was to develop an alternative approach that could overcome this obstacle. Eight-week-old low-density lipoprotein receptor-deficient (LDLR−/−) mice were transplanted with hematopoietic stem cell (HSC)-enriched bone marrow cells transduced with lentivectors expressing either green fluorescent protein (GFP) (Lenti-SP-GFP, control) or LXRα (Lenti-SP-LXRα) driven by a synthetic macrophage promoter. At 4 weeks post-transplant, the mice were fed with a Western diet for 8 weeks and then killed. Compared with Lenti-SP-GFP mice, the Lenti-SP-LXRα mice had a 30% reduction in atherosclerotic lesions, which was accompanied by increases in levels of macrophage expression of cholesterol efflux genes apolipoprotein E and ATP-binding cassette A1, as well as decreases in plasma inflammatory cytokines interleukin-6 and tumor necrosis factor-α. Intriguingly, a 50% reduction of plasma triglyceride level was also observed. We conclude that HSC-based macrophage LXRα gene therapy ameliorates the development of atherosclerosis along with an unexpected concomitant reduction of plasma triglyceride levels in LDLR−/− mice. These findings highlight the potential value of macrophage LXR expression as an avenue for therapeutic intervention against atherosclerosis.

Hematopoietic knockdown of PPARδ reduces atherosclerosis in LDLR-/- mice

PPARδ (peroxisome proliferator-activated receptor δ) mediates inflammation in response to lipid accumulation. Systemic administration of a PPARδ agonist can ameliorate atherosclerosis. Paradoxically, genetic deletion of PPARδ in hematopoietic cells led to a reduction of atherosclerosis in murine models, suggesting that downregulation of PPARδ expression in these cells may mitigate atherogenesis. To advance this finding forward to potential clinical translation through hematopoietic stem cell transplantation-based gene therapy, we employed a microRNA (miRNA) approach to knock down PPARδ expression in bone marrow cells followed by transplantation of the cells into LDLR−/− mice. We found that knockdown of PPARδ expression in the hematopoietic system caused a dramatic reduction in aortic atherosclerotic lesions. In macrophages, a key component in atherogenesis, knockdown of PPARδ led to decreased expression of multiple pro-inflammatory factors, including monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-1β and IL-6. Expression of CCR2, a receptor for MCP-1, was also decreased. The downregulation of pro-inflammatory factors is consistent with significant reduction of macrophage presence in the lesions, which may also be attributable to elevation of ABCA1 (ATP-binding cassette, subfamily A, member 1) and depression of adipocyte differentiate-related protein. Furthermore, the abundance of both MCP-1 and matrix metalloproteinase-9 proteins was reduced in plaque areas. Our results demonstrate that miRNA-mediated PPARδ knockdown in hematopoietic cells is able to ameliorate atherosclerosis.

Abstract 3521: Non-canonical activation of AR signaling by histone deacetylase SIRT1

SIRT1, a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, is a member of multigene family of Sirtuins. Traditionally SIRT1 is a known player in enhancing longevity. SIRT1 can target both histone and non-histone proteins to regulate diverse activities including cellular stress resistance, genomic stability, energy metabolism and tumorigenesis. Disruption of SIRT1 in the prostate results in the formation of prostatic intraepithelial neoplasia (PIN) lesions, suggesting a role for SIRT1 in the development of PIN, a precursor lesion for prostate cancer (PCA). Paradoxically, an oncogenic role for SIRT1 was reported as evidenced by development of micro-invasive prostate carcinomas in Pten +/- /Sirt1tg mice. Therefore, SIRT1 may possess both oncogenic and tumor suppressor activities in a context-dependent manner and its role in human cancer remains controversial. To address these paradoxical findings, we examined the role of SIRT1 in prostate pathogenesis using stably silenced for SIRT1 prostate cancer cell lines. Silencing SIRT1 in androgen responsive LNCaP cells resulted in (i) morphological changes indicative of clustering and epithelial mesenchymal transition (EMT); (ii) significant reduction in their ability to form colonies on soft agar; and (iii) increased autophagic flux. Similarly, silencing SIRT1 resulted in reduced proliferative, colony forming and migratory ability of androgen independent PC-3 cells. Furthermore, notably, genes involved in nuclear receptor (PXR/RXR/FXR/PPAR) and kinase (AMPK, MSP-RON, and RhoA) signaling are significantly altered as evidenced by RNA-seq analysis in these cells. Remarkably, SIRT1 KD cells showed decreased expression of AR and its target genes including PSA, TMPRSS-2, FKBP-5 and PMEPA1. Interestingly, SIRT1 KD reduced PSA-luciferase activity under both normal physiological and androgen deprivation growth conditions with no significant effect on nuclear or cytosolic levels of AR. More importantly, SIRT1 KD cells showed increased sensitivity to growth under androgen deprivation conditions. Intervention with resveratrol (RES, known SIRT1 activator) reduced incidence of HGPIN in prostate specific PTEN KO mouse model with no significant differences in the expression of AR or SIRT1. Consistent with these in vivo observations, SIRT1 had no significant impact on RES-induced growth inhibition. Taken together, these data indicate that SIRT1 could potentially contribute to androgen independence, independent of classical AR signaling. Our study demonstrates that SIRT1 may not play an essential role in mediating RES-induced tumor growth inhibition and that SIRT1 may confer resistance to androgen deprivation therapy and sustains cell survival, suggesting that combination of SIRT1 inhibitors and ADT might be a potential strategy for advanced PCA treatment. Supported by CPRIT RP 150166 and NCI CA 137518 (APK). Citation Format: Shih-Bo Huang, Dinesh Thapa, Suleman S. Hussain, Roble G. Bedolla, Guiming Li, Robert L. Reddick, Zhao Lai, Hung-I H. Chen, Yidong Chen, Rita Ghosh, Addanki P. Kumar. Non-canonical activation of AR signaling by histone deacetylase SIRT1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3521. doi:10.1158/1538-7445.AM2017-3521

Abstract 3673: AMPK-SIRT1 axis: a potential therapeutic target for prostate cancer management.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Recently we have shown that resveratrol (RES) intervention prevents development of high-grade prostatic intraepithelial neoplastic (HGPIN) lesions in prostate-specific PTEN knockout mouse model targeting SIRT1/mTORC1 axis. It is known that insufficient nutrient supply combined with high proliferation keeps solid tumors including prostate under hypoxic and metabolic stress. Tumor cells adapt to survive under such conditions through activation of AMP-activated kinase (AMPK). AMPK has been reported to be activated in prostate tumors. Therefore targeting AMPK and associated signaling pathways will be a promising approach for prostate cancer management. Accordingly we investigated the role of AMPK in RES-induced growth inhibitory effects using multiple human prostate cancer cell lines and preclinical animal model. These data show that RES treatment (50 μM, 24 h) results in activation of SIRT1, significant inhibition of AMPK phosphorylation and cell survival in human prostate cancer cells (RWPE-1, LNCaP, C42B and DU145). The observed molecular changes were associated with induction of apoptosis and autophagy following treatment with RES. Further dietary administration of RES (0.1 and 2%) to 4-5 week old prostate-specific PTEN knockout mice for 11 and 14 weeks showed prevention of HGPIN development. Interestingly intervention for 7 weeks showed prevention of HGPIN development at lower dose but not at high dose. On the other hand, 28-week intervention had no significant effect on the development of HGPIN lesions. Immunohistochemical evaluation showed modulation of AMPK, pS6K and SIRT1 in the prostate. Overall these data provide novel insights into RES-induced prevention of HGPIN development via AMPK/SIRT1/mTORC1 axis. Supported in part by NIH (CA 137518 and 135451 APK). Citation Format: Guiming Li, Paul Rivas, Roble Bedolla, Robert L. Reddick, Rita Ghosh, Addanki P. Kumar. AMPK-SIRT1 axis: a potential therapeutic target for prostate cancer management. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3673. doi:10.1158/1538-7445.AM2013-3673

Abstract 2585: Resveratrol induces autophagy in prostate cancer cells and intervention suppresses the progression of PIN in animals

Resveratrol (3,4′-trihydroxystilbene), a natural product is present in significant concentrations in red wine, peanuts, walnuts etc. Emerging evidence indicates that resveratrol exerts antitumorigenic activity in various tumor models including prostate. Although multiple biological effects on proliferation, apoptosis and activation of SIRT1 have been demonstrated to be associated with resveratrol-induced activities, the precise mechanism associated with resveratrol-induced SIRT1 activation and cancer cell growth inhibition remains unclear. In addition, whether resveratrol intervention suppresses the development and progression of PIN (Prostatic Intraepithelial Neoplasia) is unknown. In this study, we examined the role of SIRT1 and the underlying mechanism involved in resveratrol-mediated biological effects. Our results show that although resveratrol significantly inhibited proliferation of multiple prostate cell lines including RWPE-1, C42B, PC3 and DU145 at higher concentrations (> 50 µM); however, at lower concentrations ( −/− mice, we found that dietary administration of resveratrol reduced the incidence of high-grade PIN lesions significantly. These findings implicated an important role for mTORC1/SIRT1 signaling axis in mediating resveratrol-induced upregulation of autophagy and inhibition of apoptosis. [Supported by NIH CA137578 (APK)] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2585. doi:1538-7445.AM2012-2585