Faculty Bibliography

Aims: Glutathione (GSH) is the main antioxidant against cell damage. Several pathological states course with reduced nucleophilic tone and perturbation of redox homeostasis due to changes in the 2GSH/GSSG ratio. Here we investigated the regulation of the rate limiting GSH biosynthetic heterodimeric enzyme gamma-glutamate-cysteine- ligase (GCL) by microRNAs (miRNAs). Results: "In silico" analysis of the 3'-UTR regions of both catalytic (GCLc) and regulatory (GCLm) subunits of GCL, allowed to identify miR-433 as a strong candidate for the targeting of GCL. Transitory overexpression of miR-433 in HUVEC showed a downregulation of both GCLc and GCLm in a Nrf2-independent manner. Increases in pro-oxidant stimuli such as exposure to H2O2 or GSH depletion in endothelial and hepatic cells caused an expected increase in GCLc and GCLm protein expression and abrogation of miR-433 levels, thus supporting a cross-regulation of these pathways. Treatment of HUVEC with miR-433 resulted in reduced antioxidant and redox potentials, increased S-glutathionylation and reduced eNOS activation. In vivo models of renal and hepatic fibrosis were associated with transforming growth factor beta1 (TGF-beta1)-related reduction of GCLc and GCLm levels that were miR-433 dependent. Innovation and Conclusion: We describe for the first time a miRNA, miR-433, capable of directly targeting GCL and promoting functional consequences in endothelial physiology and fibrotic processes by decreasing GSH levels.

Marfan syndrome (MFS) is an autosomal dominant disorder of connective tissue, caused by mutations of the microfibrillar protein fibrillin-1, that predisposes affected individuals to aortic aneurysm and rupture and is associated with increased TGFbeta signaling. TGFbeta is secreted from cells as a latent complex consisting of TGFbeta, the TGFbeta propeptide, and a molecule of latent TGFbeta binding protein (LTBP). Improper extracellular localization of the latent complex can alter active TGFbeta levels, and has been hypothesized as an explanation for enhanced TGFbeta signaling observed in MFS. We previously reported the absence of LTBP-3 in matrices lacking fibrillin-1, suggesting that perturbed TGFbeta signaling in MFS might be due to defective interaction of latent TGFbeta complexes containing LTBP-3 with mutant fibrillin-1 microfibrils. To test this hypothesis, we genetically suppressed Ltbp3 expression in a mouse model of progressively severe MFS. Here, we present evidence that MFS mice lacking LTBP-3 have improved survival, essentially no aneurysms, reduced disruption and fragmentation of medial elastic fibers, and decreased Smad2/3 and Erk1/2 activation in their aortas. These data suggest that, in MFS, improper localization of latent TGFbeta complexes composed of LTBP-3 and TGFbeta contributes to aortic disease progression.

The cyclin-dependent kinase (CDK) inhibitor p27kip1 is a critical regulator of the G1/S-phase transition of the cell cycle and also regulates microtubule (MT) stability. This latter function is exerted by modulating the activity of stathmin, an MT-destabilizing protein, and by direct binding to MTs. We recently demonstrated that increased proliferation in p27kip1-null mice is reverted by concomitant deletion of stathmin in p27kip1/stathmin double-KO mice, suggesting that a CDK-independent function of p27kip1 contributes to the control of cell proliferation. Whether the regulation of MT stability by p27kip1 impinges on signaling pathway activation and contributes to the decision to enter the cell cycle is largely unknown. Here, we report that faster cell cycle entry of p27kip1-null cells was impaired by the concomitant deletion of stathmin. Using gene expression profiling coupled with bioinformatic analyses, we show that p27kip1 and stathmin conjunctly control activation of the MAPK pathway. From a molecular point of view, we observed that p27kip1, by controlling MT stability, impinges on H-Ras trafficking and ubiquitination levels, eventually restraining its full activation. Our study identifies a regulatory axis controlling the G1/S-phase transition, relying on the regulation of MT stability by p27kip1 and finely controlling the spatiotemporal activation of the Ras-MAPK signaling pathway.

During urinary tract infection (UTI), the second most common bacterial infection, dynamic interactions take place between uropathogenic E. coli (UPEC) and host urothelial cells. While significant strides have been made in the identification of the virulence factors of UPEC, our understanding of how the urothelial cells mobilize innate defenses against the invading UPEC remains rudimentary. Here we show that mouse urothelium responds to the adhesion of type 1-fimbriated UPEC by rapidly activating the canonical NF-kappaB selectively in terminally differentiated, superficial (umbrella) cells. This activation depends on a dual ligand/receptor system, one between FimH adhesin and uroplakin Ia and another between lipopolysaccharide and Toll-like receptor 4. When activated, all the nuclei (up to 11) of a multinucleated umbrella cell are affected, leading to significant amplification of proinflammatory signals. Intermediate and basal cells of the urothelium undergo NF-kappaB activation only if the umbrella cells are detached or if the UPEC persistently express type 1-fimbriae. Inhibition of NF-kappaB prevents the urothelium from clearing the intracellular bacterial communities, leading to prolonged bladder colonization by UPEC. Based on these data, we propose a model of dual ligand/receptor system in innate urothelial defenses against UPEC.

Dysregulation of amyloid-beta (Abeta) metabolism is critical for Alzheimer's disease (AD) pathogenesis. Mounting evidence suggests that apolipoprotein E (ApoE) is involved in Abeta metabolism. ATP-binding cassette transporter A1 (ABCA1) is a key regulator of ApoE lipidation, which affects Abeta levels. Therefore, identifying regulatory mechanisms of ABCA1 expression in the brain may provide new therapeutic targets for AD. Here, we demonstrate that microRNA-33 (miR-33) regulates ABCA1 and Abeta levels in the brain. Overexpression of miR-33 impaired cellular cholesterol efflux and dramatically increased extracellular Abeta levels by promoting Abeta secretion and impairing Abeta clearance in neural cells. In contrast, genetic deletion of mir-33 in mice dramatically increased ABCA1 levels and ApoE lipidation, but it decreased endogenous Abeta levels in cortex. Most importantly, pharmacological inhibition of miR-33 via antisense oligonucleotide specifically in the brain markedly decreased Abeta levels in cortex of APP/PS1 mice, representing a potential therapeutic strategy for AD. SIGNIFICANCE STATEMENT: Brain lipid metabolism, in particular Apolipoprotein E (ApoE) lipidation, is critical to Abeta metabolism and Alzheimer's disease (AD). Brain lipid metabolism is largely separated from the periphery due to blood-brain barrier and different repertoire of lipoproteins. Therefore, identifying the novel regulatory mechanism of brain lipid metabolism may provide a new therapeutic strategy for AD. Although there have been studies on brain lipid metabolism, its regulation, in particular by microRNAs, is relatively unknown. Here, we demonstrate that inhibition of microRNA-33 increases lipidation of brain ApoE and reduces Abeta levels by inducing ABCA1. We provide a unique approach for AD therapeutics to increase ApoE lipidation and reduce Abeta levels via pharmacological inhibition of microRNA in vivo.

PURPOSE: Brain metastasis is the major cause of mortality among melanoma patients. A molecular prognostic test that can reliably stratify patients at initial melanoma diagnosis by risk of developing brain metastasis may inform the clinical management of these patients. EXPERIMENTAL DESIGN: We performed a retrospective, cohort-based study analyzing genome-wide and targeted microRNA expression profiling of primary melanoma tumors of three patient cohorts (n= 92, n= 119, n= 45) with extensive clinical follow up. We used Cox regression analysis to establish a microRNA-based signature that improves the ability of the current clinicopathologic staging system to predict the development of brain metastasis. RESULTS: Our analyses identified a 4-microRNA (miR-150-5p, miR-15b-5p, miR-16-5p, and miR-374b-3p) prognostic signature that, in combination with stage, distinguished primary melanomas that metastasized to the brain from non-recurrent and non-brain-metastatic primary tumors (training cohort: C-index=81.4%, validation cohort: C-index=67.4%, independent cohort: C-index=76.9%). Corresponding Kaplan-Meier curves of high- vs. low-risk patients displayed a clear separation in brain-metastasis-free and overall survival (training: p<0.001, p<0.001, validation: p=0.033, p=0.007, independent: p=0.021, p=0.022, respectively). Finally, of the microRNA in the prognostic model, we found that the expression of a key lymphocyte miRNA, miR-150-5p, which is less abundant in primary melanomas metastatic to brain, correlated with presence of CD45+ tumor infiltrating lymphocytes. CONCLUSIONS: A prognostic assay based on the described miRNA expression signature combined with the currently used staging criteria may improve accuracy of primary melanoma patient prognoses and aid clinical management of patients, including selection for adjuvant treatment or clinical trials of adjuvant therapies.

Curly, described almost a century ago, is one of the most frequently used markers in Drosophila genetics. Despite this the molecular identity of Curly has remained obscure. Here we show that Curly mutations arise in the gene dual oxidase (duox), which encodes a reactive oxygen species (ROS) generating NADPH oxidase. Using Curly mutations and RNA interference (RNAi), we demonstrate that Duox autonomously stabilizes the wing on the last day of pupal development. Through genetic suppression studies, we identify a novel heme peroxidase, Curly Su (Cysu) that acts with Duox to form the wing. Ultrastructural analysis suggests that Duox and Cysu are required in the wing to bond and adhere the dorsal and ventral cuticle surfaces during its maturation. In Drosophila, Duox is best known for its role in the killing of pathogens by generating bactericidal ROS. Our work adds to a growing number of studies suggesting that Duox's primary function is more structural, helping to form extracellular and cuticle structures in conjunction with peroxidases.

BACKGROUND: Diabetes and aging are known risk factors for impaired neovascularization in response to ischemic insult, resulting in chronic wounds, and poor outcomes following myocardial infarction and cerebrovascular injury. Hypoxia-inducible factor (HIF)-1alpha, has been identified as a critical regulator of the response to ischemic injury and is dysfunctional in diabetic and elderly patients. To better understand the role of this master hypoxia regulator within cutaneous tissue, the authors generated and evaluated a fibroblast-specific HIF-1alpha knockout mouse model. METHODS: The authors generated floxed HIF-1 mice (HIF-1) by introducing loxP sites around exon 1 of the HIF-1 allele in C57BL/6J mice. Fibroblast-restricted HIF-1alpha knockout (FbKO) mice were generated by breeding our HIF-1 with tamoxifen-inducible Col1a2-Cre mice (Col1a2-CreER). HIF-1alpha knockout was evaluated on a DNA, RNA, and protein level. Knockout and wild-type mice were subjected to ischemic flap and wound healing models, and CD31 immunohistochemistry was performed to assess vascularity of healed wounds. RESULTS: Quantitative real-time polymerase chain reaction of FbKO skin demonstrated significantly reduced Hif1 and Vegfa expression compared with wild-type. This finding was confirmed at the protein level (p < 0.05). HIF-1alpha knockout mice showed significantly impaired revascularization of ischemic tissue and wound closure and vascularity (p < 0.05). CONCLUSIONS: Loss of HIF-1alpha from fibroblasts results in delayed wound healing, reduced wound vascularity, and significant impairment in the ischemic neovascular response. These findings provide new insight into the importance of cell-specific responses to hypoxia during cutaneous neovascularization.