Faculty Bibliography

Bone formation and skeletal repair are dynamic processes involving a fine-tuned balance between osteoblast proliferation and differentiation orchestrated by multiple signaling pathways. Canonical Wnt (cWnt) signaling is known to playing a key role in these processes. In the current study, using a transgenic mouse model with targeted disruption of axin2, a negative regulator of cWnt signaling, we investigated the impact of enhanced activation of cWnt signaling on the osteogenic capacity and skeletal repair. Specifically, we looked at two calvarial bones of different embryonic tissue origin: the neural crest-derived frontal bone and the mesoderm-derived parietal bone, and we investigated the proliferation and apoptotic activity of frontal and parietal bones and derived osteoblasts. We found dramatic differences in cell proliferation and apoptotic activity between Axin2-/- and wild type calvarial bones, with Axin2-/- showing increased proliferative activity and reduced levels of apoptosis. Furthermore, we compared osteoblast differentiation and bone regeneration in Axin2-/- and wild type neural crest-derived frontal and mesoderm-derived parietal bones, respectively. Our results demonstrate a significant increase either in osteoblast differentiation or bone regeneration in Axin2-/- mice as compared to wild type, with Axin2-/- parietal bone and derived osteoblasts displaying a "neural crest-derived frontal bone-like" profile, which is typically characterized by higher osteogenic capacity and skeletal repair than parietal bone. Taken together, our results strongly suggest that enhanced activation of cWnt signaling increases the skeletal potential of a calvarial bone of mesoderm origin, such as the parietial bone to a degree similar to that of a neural crest origin bone, like the frontal bone. Thus, providing further evidence for the central role played by the cWnt signaling in osteogenesis and skeletal-bone regeneration.

Antisense oligonucleotides (ASOs) are synthetic, single-strand RNA-DNA hybrids that induce catalytic degradation of complementary cellular RNAs via RNase H. ASOs are widely used as gene knockdown reagents in tissue culture and in Xenopus and mouse model systems. To test their effectiveness in zebrafish, we targeted 20 developmental genes and compared the morphological changes with mutant and morpholino (MO)-induced phenotypes. ASO-mediated transcript knockdown reproduced the published loss-of-function phenotypes for oep, chordin, dnd, ctnnb2, bmp7a, alk8, smad2 and smad5 in a dosage-sensitive manner. ASOs knocked down both maternal and zygotic transcripts, as well as the long noncoding RNA (lncRNA) MALAT1. ASOs were only effective within a narrow concentration range and were toxic at higher concentrations. Despite this drawback, quantitation of knockdown efficiency and the ability to degrade lncRNAs make ASOs a useful knockdown reagent in zebrafish.

Fibroblasts have a central role in wound healing via matrix production, remodeling, and contraction. Their role as mechanoresponsive cells during tissue repair is evident, but the molecular mechanisms of this process remain uncertain. Filamin A, an intracellular protein that stabilizes the actin cytoskeleton regulates fibroblast-matrix interactions. Fibroblast defects in cytoskeletal dynamics may underlie key aspects of chronic wound pathophysiology.

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.

Invariant natural killer T (iNKT) cells recognize glycolipids as antigens and diversify into NKT1 (IFN-gamma), NKT2 (IL-4), and NKT17 (IL-17) functional subsets while developing in the thymus. Mechanisms that govern the balance between these functional subsets are poorly understood due, partly, to the lack of distinguishing surface markers. Here we identify the heparan sulfate proteoglycan syndecan-1 (sdc1) as a specific marker of naive thymic NKT17 cells in mice and show that sdc1 deficiency significantly increases thymic NKT17 cells at the expense of NKT1 cells, leading to impaired iNKT cell-derived IFN-gamma, both in vitro and in vivo. Using surface expression of sdc1 to identify NKT17 cells, we confirm differential tissue localization and interstrain variability of NKT17 cells, and reveal that NKT17 cells express high levels of TCR-beta, preferentially use Vbeta8, and are more highly sensitive to a-GalCer than to CD3/CD28 stimulation. These findings provide a novel, noninvasive, simple method for identification, and viable sorting of naive NKT17 cells from unmanipulated mice, and suggest that sdc1 expression negatively regulates homeostasis in iNKT cells. In addition, these findings lay the groundwork for investigating the mechanisms by which sdc1 regulates NKT17 cells.

The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP affects protein folding homeostasis and the response to ER stress. Reversible inactivating covalent modification of BiP is believed to contribute to the balance between chaperones and unfolded ER proteins, but the nature of this modification has so far been hinted at indirectly. We report that deletion of FICD, a gene encoding an ER-localized AMPylating enzyme, abolished detectable modification of endogenous BiP enhancing ER buffering of unfolded protein stress in mammalian cells, whilst deregulated FICD activity had the opposite effect. In vitro, FICD AMPylated BiP to completion on a single residue, Thr(518). AMPylation increased, in a strictly FICD-dependent manner, as the flux of proteins entering the ER was attenuated in vivo. In vitro, Thr(518) AMPylation enhanced peptide dissociation from BiP 6-fold and abolished stimulation of ATP hydrolysis by J-domain cofactor. These findings expose the molecular basis for covalent inactivation of BiP.

SCOPE: The increased prevalence of cardiovascular diseases (CVDs) has been hypothesized to be the result of an increased exposure to a host of atherogenic environmental factors, paramount among them being unhealthy dietary habits. Long-chain n-3 polyunsaturated fatty acids (PUFAs) have been shown to have cardio protective effects, partially due to their ability to regulate gene expression. In this regard, increasing attention has been devoted to the role of miRNAs as regulators of multiple metabolic pathways whose deregulation has been associated with CVD risk. In this work we investigated whether miRNA expression was regulated by docosahexanoic acid, conjugated linoleic acid and cholesterol in Caco-2 cells. RESULTS: Among the modulated miRNAs, miR-107 was differentially expressed by all treatments and this modulation was independent of its hosting gene, panthothenate kinase 1, possibly through its own promoter, which contains binding sites for metabolically relevant transcription factors. Among the putative target genes of miR-107, we found some genes with key roles in circadian rhythm. Specifically, we demonstrated that binding of miR-107 to the circadian locomotor output cycles kaput gene results in the deregulation of the circadian rhythm of the cells. CONCLUSION: Since chronodisruption has been linked to metabolic disorders such as type 2 diabetes, atherosclerosis, obesity, and CVD, our findings suggest that miR-107 could represent a new approach for pharmacological treatment of these diseases.

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.

Genome-wide association studies (GWASs) have linked genes to various pathological traits. However, the potential contribution of regulatory noncoding RNAs, such as microRNAs (miRNAs), to a genetic predisposition to pathological conditions has remained unclear. We leveraged GWAS meta-analysis data from >188,000 individuals to identify 69 miRNAs in physical proximity to single-nucleotide polymorphisms (SNPs) associated with abnormal levels of circulating lipids. Several of these miRNAs (miR-128-1, miR-148a, miR-130b, and miR-301b) control the expression of key proteins involved in cholesterol-lipoprotein trafficking, such as the low-density lipoprotein (LDL) receptor (LDLR) and the ATP-binding cassette A1 (ABCA1) cholesterol transporter. Consistent with human liver expression data and genetic links to abnormal blood lipid levels, overexpression and antisense targeting of miR-128-1 or miR-148a in high-fat diet-fed C57BL/6J and Apoe-null mice resulted in altered hepatic expression of proteins involved in lipid trafficking and metabolism, and in modulated levels of circulating lipoprotein-cholesterol and triglycerides. Taken together, these findings support the notion that altered expression of miRNAs may contribute to abnormal blood lipid levels, predisposing individuals to human cardiometabolic disorders.

The endoplasmic reticulum (ER)-localized peroxiredoxin 4 (PRDX4) supports disulfide bond formation in eukaryotic cells lacking endoplasmic reticulum oxidase 1 (ERO1). The source of peroxide that fuels PRDX4-mediated disulfide bond formation has remained a mystery, because ERO1 is believed to be a major producer of hydrogen peroxide (H2O2) in the ER lumen. We report on a simple kinetic technique to track H2O2 equilibration between cellular compartments, suggesting that the ER is relatively isolated from cytosolic or mitochondrial H2O2 pools. Furthermore, expression of an ER-adapted catalase to degrade lumenal H2O2 attenuated PRDX4-mediated disulfide bond formation in cells lacking ERO1, whereas depletion of H2O2 in the cytosol or mitochondria had no similar effect. ER catalase did not effect the slow residual disulfide bond formation in cells lacking both ERO1 and PRDX4. These observations point to exploitation of a hitherto unrecognized lumenal source of H2O2 by PRDX4 and a parallel slow H2O2-independent pathway for disulfide formation.