Doctor of Philosophy, University of Georgia (2016)
Transforming growth factor-? (TGF-?)-induced fibroblast activation is a key pathological event during tissue fibrosis. Long noncoding RNA (lncRNA) is a class of versatile gene regulators participating in various cellular and molecular processes. However, the function of lncRNA in fibroblast activation is still poorly understood. In this study, we identified growth arrest-specific transcript 5 (GAS5) as a novel regulator for TGF-?-induced fibroblast activation. GAS5 expression was downregulated in cultured fibroblasts by TGF-? and in resident fibroblasts from bleomycin-treated skin tissues. Overexpression of GAS5 suppressed TGF-?-induced fibroblast to myofibroblast differentiation. Mechanistically, GAS5 directly bound mothers against decapentaplegic homolog 3 (Smad3) and promoted Smad3 binding to Protein phosphatase 1A (PPM1A), a Smad3 dephosphatase, and thus accelerated Smad3 dephosphorylation in TGF-?-treated fibroblasts. In addition, GAS5 inhibited fibroblast proliferation. Importantly, local delivery of GAS5 via adenoviral vector suppressed bleomycin-induced skin fibrosis in mice. Collectively, our data revealed that GAS5 suppresses fibroblast activation and fibrogenesis through inhibiting TGF-?/Smad3 signaling, which provides a rationale for an lncRNA-based therapy to treat fibrotic diseases.
View details for DOI 10.1152/ajpcell.00059.2020
View details for Web of Science ID 000551065300009
View details for PubMedID 32374674
Vascular remodeling is a pathological process following cardiovascular intervention. Vascular smooth muscle cells (VSMC) play a critical role in the vascular remodeling. Long noncoding RNAs (lncRNA) are a class of gene regulators functioning through various mechanisms in physiological and pathological conditions. By using cultured VSMC and rat carotid artery balloon injury model, we found that lncRNA growth arrest specific 5 (GAS5) serves as a negative regulator for VSMC survival in vascular remodeling. By manipulating GAS5 expression via adenoviral overexpression or short hairpin RNA knockdown, we found that GAS5 suppresses VSMC proliferation while promoting cell cycle arrest and inducing cell apoptosis. Mechanistically, GAS5 directly binds to p53 and p300, stabilizes p53-p300 interaction, and thus regulates VSMC cell survival via induction of p53-downstream target genes. Importantly, local delivery of GAS5 via adenoviral vector suppresses balloon injury-induced neointima formation along with an increased expression of p53 and apoptosis in neointimal SMCs. Our study demonstrated for the first time that GAS5 negatively impacts VSMC survival via activation the p53 pathway during vascular remodeling.
View details for DOI 10.1016/j.bbadis.2019.05.022
View details for Web of Science ID 000476965200038
View details for PubMedID 31167125
View details for PubMedCentralID PMC6663079
The kinase, LKB1, is a critical tumor suppressor in sporadic and familial human cancers, yet the mechanisms by which it suppresses tumor growth remain poorly understood. To investigate the tumor-suppressive capacity of four canonical families of Lkb1 substrates in vivo, we employed CRISPR/Cas9-mediated combinatorial genome editing in a mouse model of oncogenic Kras-driven lung adenocarcinoma. We demonstrate that members of the salt-inducible kinase (Sik) family are critical for constraining tumor development. Histological and gene expression similarities between Lkb1- and Sik-deficient tumors suggest that Siks and Lkb1 operate within the same axis. Furthermore, a gene expression signature reflecting Sik deficiency is enriched in LKB1 mutant human lung adenocarcinomas and is regulated by LKB1 in human cancer cell lines. Together, these findings reveal a key Lkb1-Sik tumor-suppressive axis and underscore the need to redirect the focus of efforts to elucidate the mechanisms through which LKB1 mediates tumor suppression.
View details for DOI 10.1158/2159-8290.CD-18-1237
View details for PubMedID 31350327
Smooth muscle cell (SMC) differentiation is essential for vascular development, and TGF-? signaling plays a critical role in this process. Although long non-coding RNAs (lncRNAs) regulate various cellular events, their functions in SMC differentiation remain largely unknown. Here, we demonstrate that the lncRNA growth arrest-specific 5 (GAS5) suppresses TGF-?/Smad3 signaling in smooth muscle cell differentiation of mesenchymal progenitor cells. We found that forced expression of GAS5 blocked, but knockdown of GAS5 increased, the expression of SMC contractile proteins. Mechanistically, GAS5 competitively bound Smad3 protein via multiple RNA Smad-binding elements (rSBEs), which prevented Smad3 from binding to SBE DNA in TGF-?-responsive SMC gene promoters, resulting in suppression of SMC marker gene transcription and, consequently, in inhibition of TGF-?/Smad3-mediated SMC differentiation. Importantly, other lncRNAs or artificially synthesized RNA molecules that contained rSBEs also effectively inhibited TGF-?/Smad3 signaling, suggesting that lncRNA-rSBE may be a general mechanism used by cells to fine-tune Smad3 activity in both basal and TGF-?-stimulated states. Taken together, our results have uncovered an lncRNA-based mechanism that modulates TGF-?/Smad3 signaling during SMC differentiation.
View details for DOI 10.1074/jbc.M117.790030
View details for PubMedID 28659340
View details for PubMedCentralID PMC5572929
Vascular smooth muscle cells (SMCs) and endothelial cells (ECs) are in close contact with blood vessels. SMC phenotypes can be altered during pathological vascular remodeling. However, how SMC phenotypes affect EC properties remains largely unknown. In this study, we found that PDGF-BB-induced synthetic SMCs suppressed EC proliferation and migration while exhibiting increased expression of anti-angiogenic factors, such as endostatin, and decreased pro-angiogenic factors, including CXC motif ligand 1 (CXCL1). Cyclopentenyl cytosine (CPEC), a CTP synthase inhibitor that has been reported previously to inhibit SMC proliferation and injury-induced neointima formation, induced SMC redifferentiation. Interestingly, CPEC-conditioned SMC culture medium promoted EC proliferation and migration because of an increase in CXCL1 along with decreased endostatin production in SMCs. Addition of recombinant endostatin protein or blockade of CXCL1 with a neutralizing antibody suppressed the EC proliferation and migration induced by CPEC-conditioned SMC medium. Mechanistically, CPEC functions as a cytosine derivate to stimulate adenosine receptors A1 and A2a, which further activate downstream cAMP and Akt signaling, leading to the phosphorylation of cAMP response element binding protein and, consequently, SMC redifferentiation. These data provided proof of a novel concept that synthetic SMC exhibits an anti-angiogenic SMC phenotype, whereas contractile SMC shows a pro-angiogenic phenotype. CPEC appears to be a potent stimulator for switching the anti-angiogenic SMC phenotype to the pro-angiogenic phenotype, which may be essential for CPEC to accelerate re-endothelialization for vascular repair during injury-induced vascular wall remodeling.
View details for DOI 10.1074/jbc.M116.741967
View details for Web of Science ID 000391571400027
View details for PubMedID 27821588
View details for PubMedCentralID PMC5207196
Macrophage phagocytosis plays an important role in host defense. The molecular mechanism, especially factors regulating the phagocytosis, however, is not completely understood. In the present study, we found that response gene to complement 32 (RGC-32) is an important regulator of phagocytosis. Although RGC-32 is induced and abundantly expressed in macrophage during monocyte-macrophage differentiation, RGC-32 appears not to be important for this process because RGC-32-deficient bone marrow progenitor can normally differentiate to macrophage. However, both peritoneal macrophages and bone marrow-derived macrophages with RGC-32 deficiency exhibit significant defects in phagocytosis, whereas RGC-32-overexpressed macrophages show increased phagocytosis. Mechanistically, RGC-32 is recruited to macrophage membrane where it promotes F-actin assembly and the formation of phagocytic cups. RGC-32 knock-out impairs F-actin assembly. RGC-32 appears to interact with PKC to regulate PKC-induced phosphorylation of F-actin cross-linking protein myristoylated alanine-rich protein kinase C substrate. Taken together, our results demonstrate for the first time that RGC-32 is a novel membrane regulator for macrophage phagocytosis.
View details for DOI 10.1074/jbc.M114.566653
View details for Web of Science ID 000341017200017
View details for PubMedID 24973210
View details for PubMedCentralID PMC4132778
The occurrence of stent thrombosis is one of the major obstacles limiting the long-term clinical efficacy of percutaneous coronary intervention. The anti-smooth muscle proliferation drugs coated on drug-eluting stents (DES) often indistinguishably block re-endothelialization, an essential step toward successful vascular repair, due to their nonspecific effect on endothelial cells (ECs). Therefore, identification of therapeutic targets that differentially regulate vascular smooth muscle cell (VSMC) and EC proliferation may lead to the development of ideal drugs for the next-generation DES. Our recent studies have shown that CTP synthase 1 (CTPS1) differentially regulates the proliferation of VSMC and EC after vascular injury. Therefore, CTPS1 inhibitors are promising agents for DES. In addition to CTPS1, other factors have also shown cell-specific effects on VSMC and/or EC proliferation and thus may become potential molecular targets for developing drugs to coat stents.
View details for DOI 10.1586/14779072.2014.866518
View details for Web of Science ID 000216649100006
View details for PubMedID 24325297
View details for PubMedCentralID PMC4358302
Accumulating evidences suggested that mitochondrial uncoupling protein 2 (UCP2) is involved in host defense in parasite infection, inflammation, and autoimmune responses. However, it remains unknown whether UCP2 is participated in the modulation of humoral immune response. Here we used quantitative PCR, ELISA, TUNEL assay, flow cytometry, etc. to study the role of UCP2 in spleen B lymphocytes during pathogen activation and obtained following results. First, UCP2 is highly expressed in splenocytes and its expression level in splenocytes is rapidly increased when the cells are activated by lipopolysaccharide (LPS) in vivo or by LPS plus cytokines in vitro. Second, in contrast to the wild type (WT) littermates, the UCP2 knockout (UCP2-KO) mice show an impaired humoral immune response when they are challenged by pathogen. Although UCP2-KO mice produce a normal level of IgM, the levels of IgGs are significantly less than those of WT littermates. Third, splenocytes from UCP2-KO mice are more susceptible to pathogen activation-induced apoptosis, and the high level of reactive oxygen species (ROS) in UCP2-KO mice may be the cause for the apoptosis. In conclusion, our study demonstrates that mitochondrial UCP2 plays a critical role in protecting splenocytes from oxidative stress-induced apoptosis during pathogen activation.
View details for DOI 10.1016/j.cellimm.2013.10.002
View details for Web of Science ID 000330205800006
View details for PubMedID 24291389
Vascular remodeling as a result of smooth muscle cell (SMC) proliferation and neointima formation is a major medical challenge in cardiovascular intervention. However, antineointima drugs often indistinguishably block re-endothelialization, an essential step toward successful vascular repair, because of their nonspecific effect on endothelial cells (ECs). The objective of this study is to identify a therapeutic target that differentially regulates SMC and EC proliferation.Using both rat balloon injury and mouse wire injury models, we identified CTP synthase 1 (CTPS1) as one of the potential targets that may be used for developing therapeutics for treating neointima-related disorders. CTPS1 was induced in proliferative SMCs in vitro and neointima SMCs in vivo. Blockade of CTPS1 expression by small hairpin RNA or activity by cyclopentenyl cytosine suppressed SMC proliferation and neointima formation. Surprisingly, cyclopentenyl cytosine had much less effect on EC proliferation. Of importance, blockade of CTPS1 in vivo sustained the re-endothelialization as a result of induction of CTP synthesis salvage pathway enzymes nucleoside-diphosphate kinase A and B in ECs. Diphosphate kinase B seemed to preserve EC proliferation via use of extracellular cytidine to synthesize CTP. Indeed, blockade of both CTPS1 and diphosphate kinase B suppressed EC proliferation in vitro and the re-endothelialization in vivo.Our study uncovered a fundamental difference in CTP biosynthesis between SMCs and ECs during vascular remodeling, which provided a novel strategy by using cyclopentenyl cytosine or other CTPS1 inhibitors to selectively block SMC proliferation without disturbing or even promoting re-endothelialization for effective vascular repair after injury.
View details for DOI 10.1161/ATVBAHA.113.301561
View details for Web of Science ID 000324891400010
View details for PubMedID 24008161
View details for PubMedCentralID PMC3852677
MicroRNAs (miRNAs) are endogenous noncoding RNAs (?22 nt) that regulate target gene expression at the post-transcriptional level in the cytoplasm. Recent discoveries of the presence of miRNAs and miRNA function-required argonaute family proteins in the cell nucleus have prompted us to hypothesize that miRNAs may also have regulatory functions in the cell nucleus. In this study, we demonstrate that mouse miR-709 is predominantly located in the nucleus of various cell types and that its nuclear localization pattern rapidly changes upon apoptotic stimuli. In the cell nucleus, miR-709 directly binds to a 19-nt miR-709 recognition element on pri-miR-15a/16-1 and prevents its processing into pre-miR-15a/16-1, leading to a suppression of miR-15a/16-1 maturation. Furthermore, nuclear miR-709 participates in the regulation of cell apoptosis through the miR-15a/16-1 pathway. In summary, the present study provides the first evidence that one miRNA can control the biogenesis of other miRNAs by directly targeting their primary transcripts in the nucleus.
View details for DOI 10.1038/cr.2011.137
View details for Web of Science ID 000300942400008
View details for PubMedID 21862971
View details for PubMedCentralID PMC3292299